Classification of "Near-patient" and "Point-of-Care" SARS-CoV-2 Nucleic Acid Amplification Test Systems and a first approach to evaluate their analytical independence of operator activities.

BACKGROUND: European legislation defines as "near-patient testing" (NPT) what is popularly and in other legislations specified as "point-of-care testing" (POCT). Systems intended for NPT/POCT use must be characterized by independence from operator activities during the analytic procedure. However, tools for evaluating this are lacking. We hypothesized that the variability of measurement results obtained from identical samples with a larger number of identical devices by different operators, expressed as the method-specific reproducibility of measurement results reported in External Quality Assessment (EQA) schemes, is an indicator for this characteristic. MATERIALS AND METHODS: Legal frameworks in the EU, the USA and Australia were evaluated about their requirements for NPT/POCT. EQA reproducibility of seven SARS-CoV-2-NAAT systems, all but one designated as "POCT", was calculated from variabilities in Ct values obtained from the respective device types in three different EQA schemes for virus genome detection. RESULTS: A matrix for characterizing test systems based on their technical complexity and the required operator competence was derived from requirements of the European In Vitro Diagnostic Regulation (IVDR) 2017/746. Good EQA reproducibility of the measurement results of the test systems investigated implies that different users in different locations have no recognizable influence on their measurement results. CONCLUSION: The fundamental suitability of test systems for NPT/POCT use according to IVDR can be easily verified using the evaluation matrix presented. EQA reproducibility is a specific characteristic indicating independence from operator activities of NPT/POCT assays. EQA reproducibility of other systems than those investigated here remains to be determined.

Advancements in COVID- 19 Testing: An in-depth overview.

COVID-19 rapidly evolved as a pandemic, killing and hospitalising millions of people, creating unprecedented hurdles for communities and health care systems worldwide. The rapidly evolving pandemic prompted the head of the World Health Organisation to deliver a critical message: "test, test, test." The response from the diagnostic industry and researchers worldwide was overwhelming, resulting in more than a thousand commercial tests available in the market worldwide. Several sampling approaches and diagnostic techniques have been employed from the early stages of the pandemic, such as SARS-CoV-2 detection by targeting the viral RNA or protein, indirectly via antibody testing, biochemical estimation, and various imaging techniques, and many are still in the various stages of development and yet to be marketed. Accurate testing techniques and appropriate sampling are the need of the hour to manage, diagnose and treat the pandemic, especially in the current crisis, where SARS-CoV-2 undergoes constant mutation, evolving into various strains, which are pretty challenging. The article discusses various testing techniques as well as screening methods for detection, treatment, and management of COVID-19 transmission, such as NAAT, PCR, isothermal detection including RT-LAMP, RPA, NASBA, RCA, SDA, NEAR, and TMA, CRISPR strategy, nanotechnology approach, metagenomic profiling, point of care tests, virus neutralization test, ELISA, biomarker estimation, utilization of imaging techniques such as CT, ultrasonography, brain MRI in COVID-19 complications, and other novel strategies including microarray methods, microfluidic methods and artificial intelligence with an emphasis on advancements in the testing strategies for the diagnosis, management, and prevention of COVID-19.

Evaluation of the Visby medical COVID-19 point of care nucleic acid amplification test.

Rapid and widespread diagnostic testing is critical to providing timely patient care and reducing transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Recently, the Visby Medical COVID-19 point of care (POC) test was granted emergency use authorization (EUA) for qualitative detection of SARS-CoV-2 nucleic acid at the point of care. We evaluated its performance characteristics using residual specimens (n = 100) collected from Mayo Clinic patients using nasopharyngeal (NP) swabs and placed in viral transport media (VTM). The same specimen was tested using both the laboratory reference method (RT-qPCR) and Visby test. The reference methods utilized included a laboratory developed test with EUA (Mayo Clinic Laboratories, Rochester, MN) using the TaqMan assay on a Roche Light Cycler 480 or a commercially available EUA platform (cobas® SARS-CoV-2; Roche Diagnostics, Indianapolis, IN). Positive, negative, and overall percent agreement between the Visby COVID-19 test and the reference method were calculated. Additionally, the limit of detection (LoD) claimed by the manufacturer (1112 copies/mL) was verified with serial dilutions of heat inactivated virus. The Visby COVID-19 test correctly identified 29/30 positive samples and 69/70 negative samples, resulting in an overall concordance of 98.0%, positive percent agreement of 96.7%, and negative percent agreement of 98.6%. The abbreviated LoD experiment showed that the analytical sensitivity of the method is as low as or lower than 500 copies/mL. Our study demonstrated that Visby COVID-19 is well-suited to address rapid SARS-CoV-2 testing needs. It has high concordance with central laboratory-based RT-qPCR methods, a low rate of invalid results, and superior analytical sensitivity to some other EUA POC devices.

Performance of saliva compared with nasopharyngeal swab for diagnosis of COVID-19 by NAAT in cross-sectional studies: Systematic review and meta-analysis.

Nucleic acid amplification testing (NAAT) is the preferred method to diagnose coronavirus disease 2019 (COVID-19). Saliva has been suggested as an alternative to nasopharyngeal swabs (NPS), but previous systematic reviews were limited by the number and types of studies available. The objective of this systematic review and meta-analysis was to assess the diagnostic performance of saliva compared with NPS for COVID-19. We searched Ovid MEDLINE, Embase, Cochrane, and Scopus databases up to 24 April 2021 for studies that directly compared paired NPS and saliva specimens taken at the time of diagnosis. Meta-analysis was performed using an exact binomial rendition of the bivariate mixed-effects regression model. Risk of bias was assessed using the QUADAS-2 tool. Of 2683 records, we included 23 studies with 25 cohorts, comprising 11,582 paired specimens. A wide variety of NAAT assays and collection methods were used. Meta-analysis gave a pooled sensitivity of 87 % (95 % CI = 83-90 %) and specificity of 99 % (95 % CI = 98-99 %). Subgroup analyses showed the highest sensitivity when the suspected individual is tested in an outpatient setting and is symptomatic. Our results support the use of saliva NAAT as an alternative to NPS NAAT for the diagnosis of COVID-19.

Integrated smart analytics of nucleic acid amplification tests via paper microfluidics and deep learning in cloud computing

Pandemics such as COVID-19 have exposed global inequalities in essential health care. Here, we proposed a novel analytics of nucleic acid amplification tests (NAATs) by combining paper microfluidics with deep learning and cloud computing. Real-time amplifications of synthesized SARS-CoV-2 RNA templates were performed in paper devices. Information pertained to on-chip reactions in time-series format were transmitted to cloud server on which deep learning (DL) models were preloaded for data analysis. DL models enable prediction of NAAT results using partly gathered real-time fluorescence data. Using information provided by the G-channel, accurate prediction can be made as early as 9 min, a 78% reduction from the conventional 40 min mark. Reaction dynamics hidden in amplification curves were effectively leveraged. Positive and negative samples can be unbiasedly and automatically distinguished. Practical utility of the approach was validated by cross-platform study using clinical datasets. Predicted clinical accuracy, sensitivity and specificity were 98.6%, 97.6% and 99.1%. Not only the approach reduced the need for the use of bulky apparatus, but also provided intelligent, distributable and robotic insights for NAAT analysis. It set a novel paradigm for analyzing NAATs, and can be combined with the most cutting-edge technologies in fields of biosensor, artificial intelligence and cloud computing to facilitate fundamental and clinical research.Copyright © 2023 Elsevier Ltd

Paper microfluidics with deep learning for portable intelligent nucleic acid amplification tests.

During global outbreaks such as COVID-19, regular nucleic acid amplification tests (NAATs) have posed unprecedented burden on hospital resources. Data of traditional NAATs are manually analyzed post assay. Integration of artificial intelligence (AI) with on-chip assays give rise to novel analytical platforms via data-driven models. Here, we combined paper microfluidics, portable optoelectronic system with deep learning for SARS-CoV-2 detection. The system was quite streamlined with low power dissipation. Pixel by pixel signals reflecting amplification of synthesized SARS-CoV-2 templates (containing ORF1ab, N and E genes) can be real-time processed. Then, the data were synchronously fed to the neural networks for early prediction analysis. Instead of the quantification cycle (Cq) based analytics, reaction dynamics hidden at the early stage of amplification curve were utilized by neural networks for predicting subsequent data. Qualitative and quantitative analysis of the 40-cycle NAATs can be achieved at the end of 22nd cycle, reducing time cost by 45%. In particular, the attention mechanism based deep learning model trained by microfluidics-generated data can be seamlessly adapted to multiple clinical datasets including readouts of SARS-CoV-2 detection. Accuracy, sensitivity and specificity of the prediction can reach up to 98.1%, 97.6% and 98.6%, respectively. The approach can be compatible with the most advanced sensing technologies and AI algorithms to inspire ample innovations in fields of fundamental research and clinical settings.

ULTRA-RAPID REAL-TIME MICROFLUIDIC POLYMERASE CHAIN REACTION INSTRUMENT FOR CLINICAL AND FORENSIC APPLICATIONS

The SARS-CoV-2 pandemic has elevated the development of novel diagnostic solutions, including rapid nucleic acid amplification tests (NAATs), to a global priority to meet the high demand for accurate, timely viral detection and diagnosis. However, ubiquitously implemented NAATs, such as polymerase chain reaction (PCR), consume hours of testing. We report a field-forward instrument capable of ultra-fast real-time PCR for amplification-based nucleic acid detection in a custom-designed microfluidic chip. Prudent selection and unconventional positioning of thermal cyclers relative to the microfluidic chip and a fluorescent detector permit ultra-fast simultaneous amplification and detection, with 40 cycles complete in under 10 minutes. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.

A collaborative study to establish the national standard for SARS-CoV-2 RNA nucleic acid amplification techniques (NAAT) in Taiwan.

The conventional PCR remains a valuable method to detect the newly emergent coronavirus rapidly and accurately. Our investigation aimed to establish the standard materials of SARS-CoV-2 for NAAT detection. We provided formalin-inactivated SARS-CoV-2 and confirmed RNA copy numbers. In addition, the virus genome was confirmed with whole-genome sequencing and identified as Wuhan/WI04/2019. Seven laboratories were invited for this collaborative study, according to the reporting data, we determined the SARS-CoV-2 with the unit of 6.35 Log10 copies/mL as the national standard. The availability of the national standard (NS) of SARS-CoV-2 will facilitate the standardization and harmonization of SARS-CoV-2 NAAT assays.

Guidance for SARS-CoV-2 RNA-Based Molecular Assay Analytical Performance Evaluations.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is frequently diagnosed through detection of viral RNA using nucleic acid amplification testing (NAAT) assays that are usually used in centralized settings. Following the publication of the SARS-CoV-2 genetic sequence, multiple diagnostic assays were launched in 2020. These assays require evaluation beyond manufacturer self-reported performance to determine whether they are suitable for use, meet country acceptance criteria, and are compatible with existing in-country platforms. In order to meet the demand for testing services, rapid yet robust assay performance evaluations are required. In our setting, these evaluation protocols required the use of residual patient specimens and reference materials, as typical clinical trials are time-consuming and limited by cost and the cyclical nature of SARS-CoV-2 infection. This protocol is designed to assist in the rapid and robust evaluation of nucleic acid-based assays for the detection of SARS-CoV-2 using limited specimens, reference materials, and test kits. While it is specific for RNA-based assays, it can be adapted for fully automated analyses. The preparation and processing of evaluation panels is described, followed by methods for analytical precision analysis and data visualization. Assay robustness and scalability are briefly discussed as these can be critical for implementation. This protocol is designed to be flexible and alternative options are provided throughout the text where possible.

Progression of LAMP as a Result of the COVID-19 Pandemic: Is PCR Finally Rivaled?

Reflecting on the past three years and the coronavirus disease 19 (COVID-19) pandemic, varying global tactics offer insights into the most effective public-health responses. In the US, specifically, rapid and widespread testing was quickly prioritized to lower restrictions sooner. Essentially, only two types of COVID-19 diagnostic tests were publicly employed during the peak pandemic: the rapid antigen test and reverse transcription polymerase chain reaction (RT-PCR). However, neither test ideally suited the situation, as rapid antigen tests are far too inaccurate, and RT-PCR tests require skilled personnel and sophisticated equipment, leading to long wait times. Loop-mediated isothermal amplification (LAMP) is another exceptionally accurate nucleic acid amplification test (NAAT) that offers far quicker time to results. However, RT-LAMP COVID-19 tests have not been embraced as extensively as rapid antigen tests or RT-PCR. This review will investigate the performance of current RT-LAMP-based COVID-19 tests and summarize the reasons behind the hesitancy to embrace RT-LAMP instead of RT-PCR. We will also look at other LAMP platforms to explore possible improvements in the accuracy and portability of LAMP, which could be applied to COVID-19 diagnostics and future public-health outbreaks.

Strategies That Facilitate Extraction-Free SARS-CoV-2 Nucleic Acid Amplification Tests.

The COVID-19 pandemic has resulted in an unprecedented global demand for in vitro diagnostic reagents. Supply shortages and hoarding have impacted testing capacity which has led to inefficient COVID-19 case identification and transmission control, predominantly in developing countries. Traditionally, RNA extraction is a prerequisite for conducting SARS-CoV-2 nucleic acid amplification tests (NAAT); however, simplified methods of sample processing have been successful at bypassing typical nucleic acid extraction steps, enabling extraction-free SARS-CoV-2 NAAT workflows. These methods involve chemical and physical approaches that are inexpensive and easily accessible alternatives to overcome extraction kit supply shortages, while offering acceptable test performance. Here we provide an overview of three main sample preparation strategies that have been shown to facilitate extraction-free SARS-CoV-2 NAATs.

A nucleic acid amplification test-based strategy does not help inform return to work for healthcare workers with COVID-19.

OBJECTIVE: The objective of this study is to assess the utility of a nucleic acid amplification test-based approach to shorten isolation of healthcare workers (HCWs) with COVID-19 in the setting of the highly transmissible omicron variant. METHODS: Between December 24, 2021, and January 5, 2022, HCWs who tested positive for SARS-CoV-2 were retested with PCR at least 5 days since onset of symptoms. RESULTS: Forty-six sequential fully COVID-19 vaccinated HCWs who had tested positive for SARS-CoV-2 underwent follow-up testing. All the samples were confirmed as omicron variants and only four (8.7%) were negative in the follow-up test performed at a median of 6 (range 5-12) since onset of symptoms. CONCLUSIONS: Implementation of a test-based strategy is logistically challenging, increases costs, and did not lead to shorter isolation in our institution.

Molecular diagnostics of SARS-CoV-2: Findings of an international survey.

BACKGROUND: In the current COVID-19 pandemic, early and rapid diagnosis of potentially infected and contagious individuals enables containment of the disease through quarantine and contact tracing. The rapid global expansion of these diagnostic testing services raises questions concerning the current state of the art with regard to standardization of testing and quality assessment practices. The aim of this study was to provide a global overview of the test methods, laboratory procedures and quality assessment practices used for SARS-CoV-2 diagnostics. METHODS: The Molecular Diagnostics Committee of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC C-MD) initiated a survey among international laboratories performing molecular genetic detection of SARS-CoV-2. Questions on quality assurance, variant testing, sequencing and the transmission of findings were included in the survey. RESULTS: A total of 273 laboratories from 49 countries participated in the survey. The majority of the participating laboratories (92.2%) use reverse transcriptase polymerase chain reaction (RT-PCR). The majority of participating laboratories do not conduct testing to identify SARS CoV-2 variants. Participation in external quality assessment programs was reported by the majority of laboratories, however, 33.2% of the laboratories reported not participating in external quality assurance programmes. CONCLUSIONS: Based on the survey, molecular diagnostic methods for SARS-CoV-2 detection are clearly not standardized across different countries and laboratories. The survey found an array of responses in regard to sample preparation, collection, processing and reporting of results. This work suggests quality assurance is insufficiently performed by diagnostic laboratories conducting SARS-CoV-2 testing.

Lung donation and SARS-CoV-2 transmission: Missed detection versus missed opportunity?

Point-of-care tests may play a valuable role in reducing the risk of donor-derived SARS-CoV-2 transmission in lung transplantation.

The One Hour COVID Test: A Rapid Colorimetric Reverse-Transcription LAMP-Based COVID-19 Test Requiring Minimal Equipment.

At this writing, over 100 million people have tested positive for Corona Virus Disease-19 (COVID-19), and the global death toll from this disease has reached nearly 3 million. Despite the many tests currently available, we have not yet achieved the testing capacity needed to limit the spread of the virus and mitigate suffering worldwide. We have developed the One Hour COVID Test to address this challenge. Our test leverages an easy-to-use, commercially available oral swab kit for sample collection paired with a novel RNA processing protocol and a simple colorimetric assay that requires minimal equipment. The test can be easily scaled via automation and takes 1 h from sample collection to result.

Comparison of Antigen Tests and qPCR in Rapid Diagnostics of Infections Caused by SARS-CoV-2 Virus.

Diagnostics of the coronavirus disease 2019 (COVID-19) using molecular techniques from the collected respiratory swab specimens requires well-equipped laboratory and qualified personnel, also it needs several hours of waiting for results and is expensive. Antigen tests appear to be faster and cheaper but their sensitivity and specificity are debatable. The aim of this study was to compare a selected antigen test with quantitative polymerase chain reaction (qPCR) tests results. Nasopharyngeal swabs were collected from 192 patients with COVID-19 symptoms. All samples were tested using Vitassay qPCR SARS-CoV-2 kit and the Humasis COVID-19 Ag Test (MedSun) antigen immunochromatographic test simultaneously. Ultimately, 189 samples were tested; 3 samples were excluded due to errors in taking swabs. The qPCR and antigen test results were as follows: 47 positive and 142 negative, and 45 positive and 144 negative, respectively. Calculated sensitivity of 91.5% and specificity of 98.6% for the antigen test shows differences which are not statistically significant in comparison to qPCR. Our study showed that effectiveness of the antigen tests in rapid laboratory diagnostics is high enough to be an alternative and support for nucleic acid amplification tests (NAAT) in the virus replication phase in the course of COVID-19.

Advances in laboratory detection methods and technology application of SARS-CoV-2.

At present, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is raging worldwide, and the coronavirus disease 2019 outbreak caused by SARS-CoV-2 seriously threatens the life and health of all humankind. There is no specific medicine for novel coronavirus yet. So, laboratory diagnoses of novel coronavirus as soon as possible and isolation of the source of infection play a vital role in preventing and controlling the epidemic. Therefore, selecting appropriate detection techniques and methods is particularly important to improve the efficiency of disease diagnosis and treatment and to curb the outbreak of infectious diseases. In this paper, virus nucleic acid, protein, and serum immunology were reviewed to provide a reference for further developing virus detection technology to provide better prevention and treatment strategies and research ideas for clinicians and researchers.

Laboratory Diagnosis of SARS-CoV-2 Pneumonia.

The emergence and rapid proliferation of Coronavirus Disease-2019, throughout the past year, has put an unprecedented strain on the global schema of health infrastructure and health economy. The time-sensitive agenda of identifying the virus in humans and delivering a vaccine to the public constituted an effort to flatten the statistical curve of viral spread as it grew exponentially. At the forefront of this effort was an exigency of developing rapid and accurate diagnostic strategies. These have emerged in various forms over the past year-each with strengths and weaknesses. To date, they fall into three categories: (1) those isolating and replicating viral RNA in patient samples from the respiratory tract (Nucleic Acid Amplification Tests; NAATs), (2) those detecting the presence of viral proteins (Rapid Antigen Tests; RATs) and serology-based exams identifying antibodies to the virus in whole blood and serum. The latter vary in their detection of immunoglobulins of known prevalence in early-stage and late-stage infection. With this review, we delineate the categories of testing measures developed to date, analyze the efficacy of collecting patient specimens from diverse regions of the respiratory tract, and present the up and coming technologies which have made pathogen identification easier and more accessible to the public.

Tools and Techniques for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)/COVID-19 Detection.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory disease coronavirus 2 (SARS-CoV-2), has led to millions of confirmed cases and deaths worldwide. Efficient diagnostic tools are in high demand, as rapid and large-scale testing plays a pivotal role in patient management and decelerating disease spread. This paper reviews current technologies used to detect SARS-CoV-2 in clinical laboratories as well as advances made for molecular, antigen-based, and immunological point-of-care testing, including recent developments in sensor and biosensor devices. The importance of the timing and type of specimen collection is discussed, along with factors such as disease prevalence, setting, and methods. Details of the mechanisms of action of the various methodologies are presented, along with their application span and known performance characteristics. Diagnostic imaging techniques and biomarkers are also covered, with an emphasis on their use for assessing COVID-19 or monitoring disease severity or complications. While the SARS-CoV-2 literature is rapidly evolving, this review highlights topics of interest that have occurred during the pandemic and the lessons learned throughout. Exploring a broad armamentarium of techniques for detecting SARS-CoV-2 will ensure continued diagnostic support for clinicians, public health, and infection prevention and control for this pandemic and provide advice for future pandemic preparedness.

Predictors of in-hospital Outcomes in Patients With Cirrhosis and Coronavirus Disease-2019.

Background: Coronavirus disease-2019 (COVID-19) cases continue to increase globally. Poor outcomes in patients with COVID-19 and cirrhosis have been reported; predictors of outcome are unclear. The existing data is from the early part of the pandemic when variants of concern (VOC) were not reported. Aims: We aimed to assess the outcomes and predictors in patients with cirrhosis and COVID-19. We also compared the differences in outcomes between the first wave of pandemic and the second wave. Methods: In this retrospective analysis of a prospectively maintained database, data on consecutive cirrhosis patients (n = 221) admitted to the COVID-19 care facility of a tertiary care center in India were evaluated for presentation, the severity of liver disease, the severity of COVID-19, and outcomes. Results: The clinical presentation included: 18 (8.1%) patients had compensated cirrhosis, 139 (62.9%) acute decompensation (AD), and 64 (29.0%) had an acute-on-chronic liver failure (ACLF). Patients with ACLF had more severe COVID-19 infection than those with compensated cirrhosis and AD (54.7% vs. 16.5% and 33.3%, P < 0.001). The overall mortality was 90 (40.7%), the highest among ACLF (72.0%). On multivariate analysis, independent predictors of mortality were high leukocyte count, alkaline phosphatase, creatinine, child class, model for end-stage liver disease (MELD) score, and COVID-19 severity. The second wave had more cases of severe COVID-19 as compared to the first wave, with a similar MELD score and Child score. The overall mortality was similar between the two waves. Conclusion: Patients with COVID-19 and cirrhosis have high mortality (40%), particularly those with ACLF (72%). A higher leukocyte count, creatinine, alkaline phosphatase, Child class, and MELD score are predictors of mortality.