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Characterizing Long Covid In An International Cohort: 7 Months Of Symptoms And Their Impact
The COVID-19 pandemic caused by SARS-CoV-2 has drawn attention to the need for fast and accurate diagnostic testing. Concerns from emerging SARS-CoV-2 variants and other circulating respiratory viral pathogens further underscore the importance of expanding diagnostic testing to multiplex detection, as single-plex diagnostic testing may fail to detect emerging variants and other viruses, while sequencing can be too slow and too expensive as a diagnostic tool. As a result, there have been significant advances in multiplex nucleic-acid-based virus diagnostic testing, creating a need for a timely review. This review first introduces frequent nucleic acid targets for multiplex virus diagnostic tests, then proceeds to a comprehensive and up-to-date overview of multiplex assays that incorporate various detection reactions and readout modalities. The performances, advantages, and disadvantages of these assays are discussed, followed by highlights of platforms that are amenable for point-of-care use. Finally, this review points out the remaining technical challenges and shares perspectives on future research and development. By examining the state of the art and synthesizing existing development in multiplex nucleic acid diagnostic tests, this review can provide a useful resource for facilitating future research and ultimately combating COVID-19.
Since the COVID-19 pandemic began in December 2019, the world has seen over 631 million confirmed cases of infection and nearly 6.6 million deaths [1]. In response, the society has galvanized into actions to combat COVID-19. For example, vaccination against the causative virus, SARS-CoV-2, was miraculously developed in one year after the onset of the pandemic, and since then, ~12.8 billion doses of vaccines have been administered worldwide [1, 2]. However, as many regions of the world still suffer from low vaccination rates—coupled with uneven public safety policies and travel restrictions across the globe—several highly transmissible SARS-CoV-2 variants have emerged and caused multiple waves during the pandemic, each time straining the healthcare system [1, 2, 3, 4]. There is also the risk of co-circulation and even co-infection with other respiratory viruses [5]. Thus, nearly 3 years into the pandemic, this global threat has persisted and evolved.
Throughout the pandemic, the need for rapid, accurate, accessible, and cost-effective diagnostic testing of SARS-CoV-2 based on its viral RNA has been in the spotlight. As the threat from SARS-CoV-2 evolves, the need for diagnostic technologies evolves accordingly, where the detection of SARS-CoV-2 mutations and variants alongside other respiratory viruses has become paramount. To this end, sequencing has been indispensable for tracking genetic mutations in SARS-CoV-2 and other respiratory viruses. The cost, complexity, and lag time of sequencing, however, render it ineffective as a timely diagnostic testing option [6, 7]. On the other hand, single-plex diagnostic testing assays that already detect SARS-CoV-2 viral RNA can be expanded to detect multiple targets. In fact, many existing SARS-CoV-2 diagnostic testing assays either detect multiple fragments from one gene or multiple genes to reduce false negative rates due to RNA degradation or false positive rates due to amplification errors [8, 9, 10, 11, 12]. Many researchers have also developed multiplexed virus diagnostic testing assays that can detect SARS-CoV-2 variants and other respiratory viruses [13, 14, 15, 16, 17]. Some researchers have further developed multiplexed virus diagnostic testing platforms that are amenable for point-of-care (POC) use [18, 19, 20]. These represent significant advances since the onset of the pandemic, and further advances can be anticipated.
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We recognize that, in addition to research, a comprehensive review that examines the state of the art and synthesizes existing development of multiplex nucleic-acid-based virus diagnostic tests can be beneficial for guiding future research, propelling further advances, and combating COVID-19. Although there are numerous reviews on various biosensing techniques and technologies for detecting SARS-CoV-2 [21, 22, 23, 24, 25], there is only one recent review on multiplex biosensing for SARS-CoV-2 mutation detection [26], and there has yet to be a review focusing on multiplex nucleic-acid-based viral diagnostic tests that also target SARS-CoV-2 variants and other respiratory viruses. In our review, we first briefly introduce the frequent targets for multiplexed virus diagnostic tests. We then provide an up-to-date overview of multiplex virus diagnostic testing assays that incorporate various reaction techniques and detection modalities (Figure 1) while commenting on their performances, advantages, and disadvantages. We also highlight multiplexed virus diagnostic tests that have been implemented within platforms that are amenable for POC use. Finally, we offer perspectives on future research. In doing so, through this review, we hope to facilitate further advances in multiplexed virus diagnostic tests for combating the persistent and evolving threat of COVID-19.
We begin our review by pointing out that we use a liberal definition of multiplex detection to ensure broad coverage of existing diagnostic tests. By traditional definition, a multiplex assay is performed within a single reaction tube (or well) and can simultaneously detect multiple targets that may all be present in a single sample. However, to ensure the breadth of our review, we define “multiplex” as any assay that detects more than one nucleic acid target. Based on this definition, we also include assays that incorporate multiple parallel independent reactions, each typically only detecting one target. These independent reactions can be performed within a single device that houses multiple reaction wells, which can essentially function as a multiplex test.
In this review, targets refer to viral RNA, including SARS-CoV-2 genes and genetic mutations, which lead to SARS-CoV-2 variants, as well as genes of other respiratory viruses. SARS-CoV-2 is an enveloped virus that consists of a lipid membrane and is developed and synthesized by host-cell machinery [3, 4]. Four key genes encode key functional proteins [5] and have been commonly targeted: the N gene that encodes the nucleocapsid (N) protein [27], the M gene that encodes the matrix (M) protein [28], the E gene that encodes the envelope (E) protein, and the S gene that encodes the spike (S) glycoprotein [29]. In addition, RNA-dependent RNA polymerase (RdRp) gene and open reading frames (ORFs) regions of the SARS-CoV-2 genome, which enable replication of N, M, E, and S proteins, can also serve as targets for SARS-CoV-2 detection. Among these targets, the N, M, and E genes are more stable throughout the evolution of SARS-CoV-2, while S, RdRp, and ORFs are more liable to undergo a mutation [30]. For example, RdRp is prone to errors due to a lack of a proofreading mechanism, causing 10
Continuous Cell Free Replication And Evolution Of Artificial Genomic Dna In A Compartmentalized Gene Expression System
Mutations per base pair [31]. Such genetic mutations, in combination with homologous recombination, make the viral diversity of SARS-CoV-2 immense and give rise to SARS-CoV-2 variants. As of late October 2022, the World Health Organization has tracked numerous variants, currently designating Omicron as a variant of concern (VOC) and monitoring its various subvariants, while listing Alpha, Beta, Gamma, Delta as previously circulating VOCs, and several other variants, such as Epsilon and Iota, as previously circulating variants of interest (VOIs). Genetic mutations in SARS-CoV-2 variants can potentially cause false negatives, further driving the need for multiplex testing. For example, as the S gene harbors frequent mutations, single-plex assays that target the S gene must be regularly validated to avoid false negative results [30]. Finally, similar symptoms are shared between SARS-CoV-2 and other respiratory viral pathogens, such as non-SARS-CoV-2 coronavirus, influenza virus, adenovirus, rhinovirus/enterovirus, and parainfluenza virus. In response, the United States Center for Diseases Control and Prevention and the Food and Drug Administration have updated the testing guidelines to co-test for SARS-CoV-2 alongside Influenza A/B. As of the writing of this review, however, simultaneous testing of SARS-CoV-2 with other respiratory viruses currently remains infrequent, typically limited to Influenza A/B and respiratory syncytial virus (RSV) [32, 33, 34, 35].
To date, researchers have reported a variety of multiplex virus diagnostic testing assays that can distinguish wild-type (WT) SARS-CoV-2 genes from VOCs, in addition to co-detection methods with other respiratory viruses. Among these viral RNA detection reactions, polymerase chain reaction (PCR) remains the most common, but isothermal reactions, particularly loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), have gained significant traction since the onset of the COVID-19 pandemic. Moreover, clustered regularly interspaced short palindromic repeats (CRISPR)-based
Mutations per base pair [31]. Such genetic mutations, in combination with homologous recombination, make the viral diversity of SARS-CoV-2 immense and give rise to SARS-CoV-2 variants. As of late October 2022, the World Health Organization has tracked numerous variants, currently designating Omicron as a variant of concern (VOC) and monitoring its various subvariants, while listing Alpha, Beta, Gamma, Delta as previously circulating VOCs, and several other variants, such as Epsilon and Iota, as previously circulating variants of interest (VOIs). Genetic mutations in SARS-CoV-2 variants can potentially cause false negatives, further driving the need for multiplex testing. For example, as the S gene harbors frequent mutations, single-plex assays that target the S gene must be regularly validated to avoid false negative results [30]. Finally, similar symptoms are shared between SARS-CoV-2 and other respiratory viral pathogens, such as non-SARS-CoV-2 coronavirus, influenza virus, adenovirus, rhinovirus/enterovirus, and parainfluenza virus. In response, the United States Center for Diseases Control and Prevention and the Food and Drug Administration have updated the testing guidelines to co-test for SARS-CoV-2 alongside Influenza A/B. As of the writing of this review, however, simultaneous testing of SARS-CoV-2 with other respiratory viruses currently remains infrequent, typically limited to Influenza A/B and respiratory syncytial virus (RSV) [32, 33, 34, 35].
To date, researchers have reported a variety of multiplex virus diagnostic testing assays that can distinguish wild-type (WT) SARS-CoV-2 genes from VOCs, in addition to co-detection methods with other respiratory viruses. Among these viral RNA detection reactions, polymerase chain reaction (PCR) remains the most common, but isothermal reactions, particularly loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), have gained significant traction since the onset of the COVID-19 pandemic. Moreover, clustered regularly interspaced short palindromic repeats (CRISPR)-based
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