A comprehensive review and clinical guide to molecular and serological diagnostic tests and future development: In vitro diagnostic testing for COVID-19

COVID-19 is a contagious syndrome caused by SARS Coronavirus 2 (SARS-CoV-2) that requires rapid diagnostic testing to identify and manage in the affected persons, characterize epidemiology, and promptly make public health decisions and manage the virus present in the affected person and promptly make public health decisions by characterizing the epidemiology. Technical problems, especially contamination occurring during manual real-time polymerase chain reaction (RT-PCR), can result in false-positive NAAT results. In some cases, RNA detection technology and antigen testing are alternatives to RT-PCR. Sequencing is vital for tracking the SARS-CoV-2 genome's evolution, while antibody testing is beneficial for epidemiology. SARS-CoV-2 testing can be made safer, faster, and easier without losing accuracy. Continued technological advancements, including smartphone integration, will help in the current epidemic and prepare for the next. Nanotechnology-enabled progress in the health sector has aided disease and pandemic management at an early stage. These nanotechnology-based analytical tools can be used to quickly diagnose COVID-19. The SPOT system is used to diagnose the coronavirus quickly, sensibly, accurately, and with portability. The SPOT assay consists of RT-LAMP, followed by pfAgo-based target sequence detection. In addition, SPOT system was used to detect both positive and negative SARS-CoV-2 samples. This combination of speed, precision, sensitivity, and mobility will allow for cost-effective and high-volume COVID-19 testing. © 2023 the author(s), published by De Gruyter.

Impact of the loss of Laboratory Developed Mass Spectrometry testing at a major academic medical center.

Background: Our laboratory historically performed immunosuppressant and definitive opioid testing in-house as laboratory developed (LDT) mass spectrometry-based tests. However, staffing constraints and supply chain challenges associated with the COVID-19 pandemic forced us to refer this testing to a national reference laboratory. The VALID Act could impose onerous requirements for laboratories to develop LDTs. To explore the potential effect of these additional regulatory hurdles, we used the loss of our own LDT tests to assess the impact on patient care and hospital budgets. Methods: Laboratory information systems data and historical data associated with test costs were used to calculate turnaround times and financial impact. Results: Referral testing has extended the reporting of immunosuppressant results by an average of approximately one day and up to two days at the 95th percentile. We estimate that discontinuing in-house opioid testing has cost our health system over half a million dollars in the year since testing was discontinued. Conclusions: Barriers that discourage laboratories from developing in-house testing, particularly in the absence of FDA-cleared alternatives, can be expected to have a detrimental effect on patient care and hospital finances.

SARS-CoV-2 Virus-like Particles (VLPs) Specifically Detect Humoral Immune Reactions in an ELISA-Based Platform.

A key in controlling the SARS-CoV-2 pandemic is the assessment of the immune status of the population. We explored the utility of SARS-CoV-2 virus-like particles (VLPs) as antigens to detect specific humoral immune reactions in an enzyme-linked immunosorbent assay (ELISA). For this purpose, SARS-CoV-2 VLPs were produced from an engineered cell line and characterized by Western blot, ELISA, and nanoparticle tracking analysis. Subsequently, we collected 42 serum samples from before the pandemic (2014), 89 samples from healthy subjects, and 38 samples from vaccinated subjects. Seventeen samples were collected less than three weeks after infection, and forty-four samples more than three weeks after infection. All serum samples were characterized for their reactivity with VLPs and the SARS-CoV-2 N- and S-protein. Finally, we compared the performance of the VLP-based ELISA with a certified in vitro diagnostic device (IVD). In the applied set of samples, we determined a sensitivity of 95.5% and a specificity of 100% for the certified IVD. There were seven samples with an uncertain outcome. Our VLP-ELISA demonstrated a superior performance, with a sensitivity of 97.5%, a specificity of 100%, and only three uncertain outcomes. This result warrants further research to develop a certified IVD based on SARS-CoV-2 VLPs as an antigen.

Analysis of the renewed European Medical Device Regulations in the frame of the non - EU regulatory landscape during the COVID facilitated change.

The term "Medical devices" includes technology-based devices or articles, both basic and complex. Due to these types of variations, a strict, robust, transparent, and sustainable regulatory framework is required. In recent clinical practice, incidents including the breast implant and the hip replacement crisis have made it necessary to improve the regulatory and compliance approaches for the industry to ensure the manufacturing and distribution of safe and innovative MDs within the EU. In response to this, the EU revised the laws governing medical devices and in vitro diagnostics to align with the developments of the sector, address critical safety issues and support innovation. The new regulation (EU) 2017/745 on Medical Devices (MDR) is now applicable from May 26 2021 and the In Vitro Diagnostic Medical Devices Regulation (EU) 2017/746 will take effect from May 2022.In this review, we aim to provide an update on the new Medical Device Regulations in the context of the current medical needs of the world, and also to give a glimpse at the non-EU regulatory landscape. Finally, we take a look at the closed-system transfer devices (CSTD) and COVID facilitated changes promoting demand for continuous improvement and trends in the pharmaceutical and medical industry related areas.

Development of a Multiplex Tandem PCR (MT-PCR) Assay for the Detection of Emerging SARS-CoV-2 Variants.

The emergence of variants of SARS-CoV-2 has created challenges for the testing infrastructure. Although large-scale genome sequencing of SARS-CoV-2 has facilitated hospital and public health responses, access to sequencing facilities globally is variable and turnaround times can be significant, so there is a requirement for rapid and cost-effective alternatives. Applying a polymerase chain reaction (PCR)-based single nucleotide polymorphism (SNP) approach enables rapid (<4 h) identification of SARS-CoV-2 lineages from nucleic acid extracts, through the presence or absence of a panel of defined of genomic polymorphisms. For example, the B.1.1.7 lineage ("UK", "Alpha", or "Kent" variant) is characterised by 23 mutations compared to the reference strain, and the most biologically significant of these are found in the S gene. We have developed a SARS-CoV-2 typing assay focused on five positions in the S gene (HV69/70, N501, K417, E484 and P681). This configuration can identify a range of variants, including all the "Variants of Concern" currently designated by national and international public health bodies. The panel has been evaluated using a range of clinical isolates and standardised control materials at four UK hospitals and shows excellent concordance with the known lineage information derived from full sequence analysis. The assay has a turnaround time of about three hours for a set of up to 24 samples and has been utilised to identify emerging variants in a clinical setting.

A Novel Rapid Test to Detect Anti-SARS-CoV-2 N Protein IgG Based on Shear Horizontal Surface Acoustic Wave (SH-SAW).

Since the Coronavirus disease 2019 (COVID-19) pandemic outbreak, many methods have been used to detect antigens or antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including viral culture, nucleic acid test, and immunoassay. The shear-horizontal surface acoustic wave (SH-SAW) biosensor is a novel pathogen detection platform with the advantages of high sensitivity and short detection time. The objective of this study is to develop a SH-SAW biosensor to detect the anti-SARS-CoV-2 nucleocapsid antibody. The rabbit sera collected from rabbits on different days after SARS-CoV-2 N protein injection were evaluated by SH-SAW biosensor and enzyme-linked immunosorbent assay (ELISA). The results showed that the SH-SAW biosensor achieved a high correlation coefficient (R = 0.9997) with different concentrations (34.375-1100 ng/mL) of the "spike-in" anti-N protein antibodies. Compared to ELISA, the SH-SAW biosensor has better sensitivity and can detect anti-N protein IgG signals earlier than ELISA on day 6 (p < 0.05). Overall, in this study, we demonstrated that the SH-SAW biosensor is a promising platform for rapid in vitro diagnostic (IVD) testing, especially for antigen or antibody testing.

COVIDIAGNOSTIX: health technology assessment of serological tests for SARS-CoV-2 infection.

OBJECTIVE: In vitro diagnostic tests for SARS-COV-2, also known as serological tests, have rapidly spread. However, to date, mostly single-center technical and diagnostic performance's assessments have been carried out without an intralaboratory validation process and a health technology assessment (HTA) systematic approach. Therefore, the rapid HTA for evaluating antibody tests for SARS-COV-2 was applied. METHODS: The use of rapid HTA is an opportunity to test innovative technology. Unlike traditional HTA (which evaluates the benefits of new technologies after being tested in clinical trials or have been applied in practice for some time), the rapid HTA is performed during the early stages of developing new technology. A multidisciplinary team conducted the rapid HTA following the HTA Core Model® (version 3.0) developed by the European Network for Health Technology Assessment. RESULTS: The three methodological and analytical steps used in the HTA applied to the evaluation of antibody tests for SARS-COV-2 are reported: the selection of the tests to be evaluated; the research and collection of information to support the adoption and appropriateness of the technology; and the preparation of the final reports and their dissemination. Finally, the rapid HTA of serological tests for SARS-CoV-2 is summarized in a report that allows its dissemination and communication. CONCLUSIONS: The rapid-HTA evaluation method, in addition to highlighting the characteristics that differentiate the tests from each other, guarantees a timely and appropriate evaluation, becoming a tool to create a direct link between science and health management.

Metabolomic Laboratory-Developed Tests: Current Status and Perspectives.

Laboratory-developed tests (LDTs) are a subset of in vitro diagnostic devices, which the US Food and Drug Administration defines as "tests that are manufactured by and used within a single laboratory". The review describes the emergence and history of LDTs. The current state and development prospects of LDTs based on metabolomics are analyzed. By comparing LDTs with the scientific metabolomics study of human bio samples, the characteristic features of metabolomic LDT are shown, revealing its essence, strengths, and limitations. The possibilities for further developments and scaling of metabolomic LDTs and their potential significance for healthcare are discussed. The legal aspects of LDT regulation in the United States, European Union, and Singapore, demonstrating different approaches to this issue, are also provided. Based on the data presented in the review, recommendations were made on the feasibility and ways of further introducing metabolomic LDTs into practice.

MedTech Forum 2021 (April 20-22, 2021 - Virtual Meeting).

The MedTech Forum 2021 was an online event at which participants from the field of medical devices, diagnostics and digital health openly shared their views on the latest trends, challenges and opportunities currently being faced. The event enticed representatives from around 30 companies from the healthcare sector, as well as those from competent authorities, notified bodies, healthcare providers, academia and patients. Parallel sessions distributed across 3 days enabled the audience to interact with the speakers through a live-chat platform. Various sessions covering health data space, digitalization and interoperability were held in a forum and were a highlight of the meeting due to the potential of health data for the improvement of patient management and healthcare systems. Hot topics included the imminent application of the Medical Device Regulation (MDR) in May 2021 and the application of the In Vitro Diagnostic Device Regulation (IVDR) by May 2022. European competitiveness in the healthcare sector, learnings from the COVID-19 crisis and how to make legislation more friendly with innovations were also topics widely discussed during the event. This report provides a summary of sessions on health digitalization, the application of the new European regulations and lessons learnt from COVID-19.

The diagnostic accuracy of seven commercial molecular in vitro SARS-CoV-2 detection tests: a rapid meta-analysis.

OBJECTIVE: To compare the accuracy parameters of seven commercial molecular in vitro diagnostic tests for detecting SARS-CoV-2. METHODS: Studies evaluating the accuracy of seven different commercial molecular diagnostic tests for detecting SARS-CoV-2 (Cepheid Xpert Xpress SARS-CoV-2 test, Simplexa COVID-19 Direct, Abbott ID NOW COVID-19, Cobas SARS-CoV-2, Allplex 2019-nCoV Assay, Panther Fusion SARS-CoV-2, and BioFire COVID-19 Test) were included. The quality of the included studies was assessed using the QUADAS-2 checklist. A bivariate random-effects regression model was implemented. RESULTS: Meta-analysis of 12 included studies showed that the performances of commercial COVID-19 molecular in vitro diagnostic tests were high, with a summary sensitivity of 95.9% (95% CI 93.9-97.2%, I2 = 60.22%) and specificity of 97.2% (95% CI 95.5-98.3%, I2 = 56.66%). Among seven evaluated tests, the Abbott ID NOW COVID-19 and Simplexa COVID-19 Direct displayed lower sensitivity (91.6%, 95% CI 80.5-96.6% and 92%, 95% CI 86.2-95.5, respectively). CONCLUSION: All evaluated tests showed good accuracy. However, the slightly lower sensitivity observed in the Abbott ID Now COVID-19 and Simplexa COVID-19 Direct should be considered when deciding on a test platform. Moreover, the diagnostic accuracy of COVID-19 commercial diagnostic tests should be weighed against their ease of use and speed.

Type-I Interferon assessment in 45 minutes using the FilmArray® PCR platform in SARS-CoV-2 and other viral infections.

Low concentrations of type-I interferon (IFN) in blood seem to be associated with more severe forms of Coronavirus disease 2019 (COVID-19). However, following the type-I interferon response (IR) in early stage disease is a major challenge. We evaluated detection of a molecular interferon signature on a FilmArray® system, which includes PCR assays for four interferon stimulated genes. We analyzed three types of patient populations: (i) children admitted to a pediatric emergency unit for fever and suspected infection, (ii) ICU-admitted patients with severe COVID-19, and (iii) healthcare workers with mild COVID-19. The results were compared to the reference tools, that is, molecular signature assessed with Nanostring® and IFN-α2 quantification by SIMOA® (Single MOlecule Array). A strong correlation was observed between the IR measured by the FilmArray®, Nanostring®, and SIMOA® platforms (r-Spearman 0.996 and 0.838, respectively). The FilmArray® panel could be used in the COVID-19 pandemic to evaluate the IR in 45-min with 2 min hand-on-time at hospitalization and to monitor the IR in future clinical trials.

Targets and assay types for COVID-19 diagnosis.

The lack of complete information on the immune response dynamics to infection with severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) has led to the use of mainly molecular tests such as reverse transcription PCR (RT-PCR) to diagnose Coronavirus 2019 disease (COVID-19). Although remarkable progress has been made in developing effective RT-PCR kits, the lack of specific equipment required to perform this technique in all clinical laboratories limits its widespread use. In the case of COVID-19, these tests can be used for the triage of symptomatic patients, for testing the contacts of confirmed cases, and also for the analysis and monitoring of the situation. Along with molecular tests involving reverse transcription PCR, various laboratory tests can identify the specific anti-viral antibodies or viral antigens. This review seeks to describe the targets and diagnostic methods available or currently in development for SARS-CoV-2 infection, including reverse transcription PCR (RT-PCR), serologic immunoassays (SIA) and the protein microarray method (PMM). Knowing the specific targets and the sensitivity of each assay used for COVID-19 diagnosis can lead to more efficient detection of infected patients and it can provide better management of the pandemic status.

Molecular and Serological Tests for COVID-19 a Comparative Review of SARS-CoV-2 Coronavirus Laboratory and Point-of-Care Diagnostics.

Validated and accurate laboratory testing for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a crucial part of the timely management of Coronavirus Disease 2019 (COVID-19) disease, supporting the clinical decision-making process for infection control at the healthcare level and detecting asymptomatic cases. This would facilitate an appropriate treatment, a prompt isolation and consequently deceleration of the pandemic. Various laboratory tests can identify the genetic material of SARS-CoV-2 that causes COVID-19 in specimens, or specific anti-viral antibodies in blood/serum. Due to the current pandemic situation, a development of point-of-care diagnostics (POCD) allows us to substantially accelerate taking clinical decisions and implement strategic planning at the national level of preventative measures. This review summarizes and compares the available POCD and those currently under development, including quantitative reverse transcription PCR (RT-qPCR), serology immunoassays (SIAs) and protein microarray method (PMM) designed for standard and rapid COVID-19 diagnosis.