what types of rapid testing for Covid-19 have been developed?

We present an example of applying 'need-driven' product design principle to the development of a rapid test kit to detect SARS-COV-2 (COVID-19). The tests are intended for use in the field and, longer term, for home use. They detect whether a subject is currently infected with the virus and is infectious. The urgent need for large numbers of tests in field setting imposes constraints such as short test time and lack of access to specialist equipment, laboratories and skilled technicians to perform the test and interpret results. To meet these needs, an antigen test based on RT-LAMP with colorimetric readout was chosen. Direct use of swab sample with no RNA extraction was explored. After extensive experimental study (reported elsewhere), a rapid test kit has been fabricated to satisfy all design criteria.

Development of a rapid test kit for SARS-CoV-2: an example of product design

We present an example of applying ‘need-driven’ product design principle to the development of a rapid test kit to detect SARS-COV-2 (COVID-19). The tests are intended for use in the field and, longer term, for home use. They detect whether a subject is currently infected with the virus and is infectious. The urgent need for large numbers of tests in field setting imposes constraints such as short test time and lack of access to specialist equipment, laboratories and skilled technicians to perform the test and interpret results. To meet these needs, an antigen test based on RT-LAMP with colorimetric readout was chosen. Direct use of swab sample with no RNA extraction was explored. After extensive experimental study (reported elsewhere), a rapid test kit has been fabricated to satisfy all design criteria.

In January, Mologic, embarked on a product development pathway for COVID-19 diagnostics focusing on ELISA and rapid diagnostic tests (RDTs), with anticipated funding from Wellcome Trust and DFID. 755 clinical samples from known COVID-19 patients and hospital negative controls were tested on Mologics IgG ELISA. The reported sensitivity on 191 SGUL prospectively enrolled patients was 95% on day 7 or more post diagnosis, and 97% 10 days or more post-diagnosis. A specificity panel comprising 564 samples pre-December 2019 were tested to include most common respiratory pathogens, other types of coronavirus, and flaviviruses. Specificity in this panel was 97%. This is the first in a series of Mologic products for COVID-19, which will be deployed for COVID-19 diagnosis, contact tracing and sero-epidemiological studies to estimate disease burden and transmission with a focus on ensuring access, affordability, and availability to lowest resource settings.

Rapid development of COVID-19 rapid diagnostics for low resource settings: accelerating delivery through transparency, responsiveness, and open collaboration

Fast and widespread diagnosis is crucial to fighting against the outbreak of COVID-19. This work surveys the landscape of available and emerging biosensor technologies for COVID-19 testing. Molecular diagnostic assays based on quantitative reverse transcription polymerase chain reaction are used in most clinical laboratories. However, the COVID-19 pandemic has overwhelmed testing capacity and motivated the development of fast point-of-care tests and the adoption of isothermal DNA amplification. Antigenic and serological rapid tests based on lateral-flow immunoassays suffer from low sensitivity. Advanced digital systems enhance performance at the expense of speed and the need for large equipment. Emerging technologies, including CRISPR gene-editing tools, benefit from high sensitivity and specificity of molecular diagnostics and the easy use of lateral-flow assays. DNA sequencing and sample pooling strategies are highlighted to bring out the full capacity of the available biosensor technologies and accelerate mass testing.

Trends and Innovations in Biosensors for COVID-19 Mass Testing

Fast and widespread diagnosis is crucial to fight against the outbreak of COVID-19. The present work surveys the landscape of available and emerging biosensor technologies for COVID-19 testing. Molecular diagnostic assays based on quantitative Reverse Transcription Polymerase Chain Reaction are used in most clinical laboratories. The COVID-19 pandemic has overwhelmed the testing capacity and motivated the development of fast point-of-care tests and the adoption of isothermal DNA amplification. Antigenic and serological rapid tests based on lateral flow immunoassays suffer from low sensitivity. Advanced digital systems enhance the performance at the expense of speed and large equipment requirement. Emerging technologies, including CRISPR gene-editing tools, benefit from high sensitivity and selectivity of molecular diagnostics and the easy use of lateral flow assays. DNA sequencing and sample pooling strategies are highlighted to bring out the full capacity of the available biosensor technologies and accelerate mass testing.

Trends and innovations in biosensors for COVID-19 mass testing.

Novel Corona virus (COVID-19 or 2019-nCoV) is an emerging global health concern that requires a rapid diagnostic test. Quantitative reverse transcription PCR (qRT-PCR) is currently the standard for COVID-19 detection; however, Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) may allow for faster and cheaper field based testing at point-of-risk. The objective of this study was to develop a rapid screening diagnostic test that could be completed in under 30 minutes. Simulated patient samples were generated by spiking serum, urine, saliva, oropharyngeal swabs, and nasopharyngeal swabs with a portion of the COVID-19 nucleic sequence. The samples were tested using RT-LAMP as well as by conventional qRT-PCR. Specificity of the RT-LAMP was evaluated by also testing against other related coronaviruses. RT-LAMP specifically detected COVID-19 in simulated patient samples. This test was performed in under 30 minutes. This approach could be used for monitoring of exposed individuals or potentially aid with screening efforts in the field and potential ports of entry.

Rapid Detection of Novel Coronavirus (COVID-19) by Reverse Transcription-Loop-Mediated Isothermal Amplification

There is widespread agreement that reliable, fast, and easy-to-produce diagnostic testing methods that have high sensitivity and specificity are essential for guiding appropriate responses to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak. At the present time, there are important unanswered questions about testing methods for SARS-CoV-2. This review article interprets recent findings related to the principal testing methods used to diagnose SARS-CoV-2, including reverse-transcription polymerase chain reaction (RT-PCR), chest imaging, and immunoassay. We discuss the value and limitations of these approaches and suggest directions for future research that can advance the understanding of diagnostic methods. Addressing areas of uncertainty will improve clinical outcomes and allow more effective policies to be implemented to control the disease.

What Do We Need to Know to Improve Diagnostic Testing Methods for the 2019 Novel Coronavirus?

In the last few months, an unprecedented number of laboratory tests for coronavirus disease 2019 (COVID-19) have been developed at a remarkable speed. With the rapid adoption of these tests into clinical practice, combined with the widespread publicity they received, questions arose related to the different types of tests, their utility, performance, and regulatory approval status. The aim of this publication is to provide a general landscape of laboratory testing for COVID-19 and offer a historical and regulatory perspective associated with them. Specifically, we aim to elaborate on the regulatory complexities of diagnostic testing in the United States and its implications to the present outbreak, as well as provide a synopsis of laboratory tests that have been developed for COVID-19. We will first address the detection of severe acute respiratory syndrome-coronavirus 2 directly by either nucleic acid amplification tests or by the detection of the viral protein for active infections. Subsequently, we will provide an overview of serological tests that can aid not only in diagnosis but additionally help to identify prior infections and potential immunity.

Testing For SARS-CoV-2: The Day the World Turned its Attention to the Clinical Laboratory

Clinical laboratory testing routinely provides actionable results, which help direct patient care in the inpatient and outpatient settings. Since December 2019, a novel coronavirus (SARS-CoV-2) has been causing disease (COVID-19 [coronavirus disease 2019]) in patients, beginning in China and now extending worldwide. In this context of a novel viral pandemic, clinical laboratories have developed multiple novel assays for SARS-CoV-2 diagnosis and for managing patients afflicted with this illness. These include molecular and serologic-based tests, some with point-of-care testing capabilities. Herein, we present an overview of the types of testing available for managing patients with COVID-19, as well as for screening of potential plasma donors who have recovered from COVID-19.

Types of Assays for SARS-CoV-2 Testing: A Review.

Types of Assays for SARS-CoV-2 Testing: A Review

Novel Corona virus/Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2 or 2019-nCoV), and the subsequent disease caused by the virus (coronavirus disease 2019 or COVID-19), is an emerging global health concern that requires a rapid diagnostic test. Quantitative reverse transcription PCR (qRT-PCR) is currently the standard for SARS-CoV-2 detection; however, Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) may allow for faster and cheaper field based testing at point-of-risk. The objective of this study was to develop a rapid screening diagnostic test that could be completed in 30-45 minutes. Simulated patient samples were generated by spiking serum, urine, saliva, oropharyngeal swabs, and nasopharyngeal swabs with a portion of the SARS-CoV-2 nucleic sequence. RNA isolated from nasopharyngeal swabs collected from actual COVID-19 patients was also tested. The samples were tested using RT-LAMP as well as by conventional qRT-PCR. Specificity of the RT-LAMP was evaluated by also testing against other related coronaviruses. RT-LAMP specifically detected SARS-CoV-2 in both simulated patient samples and clinical specimens. This test was performed in 30-45 minutes. This approach could be used for monitoring of exposed individuals or potentially aid with screening efforts in the field and potential ports of entry.

Rapid detection of novel coronavirus/Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) by reverse transcription-loop-mediated isothermal amplification

Novel Corona virus/Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2 or 2019-nCoV), and the subsequent disease caused by the virus (coronavirus disease 2019 or COVID-19), is an emerging global health concern that requires a rapid diagnostic test. Quantitative reverse transcription PCR (qRT-PCR) is currently the standard for SARS-CoV-2 detection; however, Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) may allow for faster and cheaper field based testing at point-of-risk. The objective of this study was to develop a rapid screening diagnostic test that could be completed in 30–45 minutes. Simulated patient samples were generated by spiking serum, urine, saliva, oropharyngeal swabs, and nasopharyngeal swabs with a portion of the SARS-CoV-2 nucleic sequence. RNA isolated from nasopharyngeal swabs collected from actual COVID-19 patients was also tested. The samples were tested using RT-LAMP as well as by conventional qRT-PCR. Specificity of the RT-LAMP was evaluated by also testing against other related coronaviruses. RT-LAMP specifically detected SARS-CoV-2 in both simulated patient samples and clinical specimens. This test was performed in 30–45 minutes. This approach could be used for monitoring of exposed individuals or potentially aid with screening efforts in the field and potential ports of entry.

Objectives SARS-CoV-2, causing COVID-19, has emerged to cause a human pandemic. Detection of SARS-CoV-2 in respiratory samples by using PCR is the standard laboratory diagnostic tool. Our aim was to perform a limited evaluation of the diagnostic performance and user-friendliness of eleven rapid tests for detection of antibodies against SARS-CoV-2. Methods All participants were tested with PCR against SARS-CoV-2 at a clinical microbiology laboratory. Comparing with results from PCR tests, we evaluated the rapid tests' performances in three arms; 1) 20 hospitalized patients with PCR-confirmed COVID-19, 2) 23 recovered outpatients with former PCR-confirmed COVID-19, and 3) 49 participants with suspected COVID-19 presenting at a primary care emergency room. Results All eleven tests detected antibodies in hospitalized COVID-19 patients, though with varying sensitivities. In former outpatients recovered from COVID-19, there were differences between tests in the immunoglobulin type G (IgG) sensitivity, with five tests having a sensitivity below 65%. In participants with suspected COVID-19 infection, the rapid tests had very low sensitivities. Most rapid tests were easy to perform and interpret. Conclusions Rapid tests were not suited as stand-alone tests to detect present infection in a Norwegian primary care emergency room population. All the rapid tests were able to detect SARS-CoV-2 antibodies, although sensitivities varied and were generally higher in the study arm of more severely affected participants. Rapid tests with high IgG sensitivity (and specificity) may be useful for confirmation of past infection. An independent evaluation should be performed in the intended population before introducing a rapid test.

Evaluation of eleven rapid tests for detection of antibodies against SARS-CoV-2.

SARS-CoV-2 is responsible for a highly contagious infection, known as COVID-19. SARS-CoV-2 was discovered in late December 2019 and, since then, has become a global pandemic. Timely and accurate COVID-19 laboratory testing is an essential step in the management of the COVID-19 outbreak. To date, assays based on the reverse-transcription polymerase chain reaction (RT-PCR) in respiratory samples are the gold standard for COVID-19 diagnosis. Unfortunately, RT-PCR has several practical limitations. Consequently, alternative diagnostic methods are urgently required, both for alleviating the pressure on laboratories and healthcare facilities and for expanding testing capacity to enable large-scale screening and ensure a timely therapeutic intervention. To date, few studies have been conducted concerning the potential utilization of rapid testing for COVID-19, with some conflicting results. Therefore, the present systematic review and meta-analysis was undertaken to explore the feasibility of rapid diagnostic tests in the management of the COVID-19 outbreak. Based on ten studies, we computed a pooled sensitivity of 64.8% (95%CI 54.5-74.0), and specificity of 98.0% (95%CI 95.8-99.0), with high heterogeneity and risk of reporting bias. We can conclude that: (1) rapid diagnostic tests for COVID-19 are necessary, but should be adequately sensitive and specific; (2) few studies have been carried out to date; (3) the studies included are characterized by low numbers and low sample power, and (4) in light of these results, the use of available tests is currently questionable for clinical purposes and cannot substitute other more reliable molecular tests, such as assays based on RT-PCR.

Point-of-Care Diagnostic Tests for Detecting SARS-CoV-2 Antibodies: A Systematic Review and Meta-Analysis of Real-World Data

Quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay is the gold standard recommended to test for acute SARS-CoV-2 infection. It has been used by the Centers for Disease Control and Prevention (CDC) and several other companies in their Emergency Use Authorization (EUA) assays. RT-qPCR requires expensive equipment such as RNA isolation instruments and real-time PCR thermal cyclers, which are not available in many low resource settings and developing countries. As a pandemic, COVID-19 has quickly spread to the rest of the world. Many underdeveloped and developing counties do not have the means for fast and accurate COVID-19 detection to control this outbreak. Using COVID-19 positive clinical specimens, we demonstrated that RT-PCR assays can be performed in as little as 12 minutes using untreated samples, heat-inactivated samples, or extracted RNA templates. Rapid RT-PCR was achieved using thin-walled PCR tubes and a setup including sous vide immersion heaters/circulators. Our data suggest that rapid RT-PCR can be implemented for sensitive and specific molecular diagnosis of COVID-19 in situations where sophisticated laboratory instruments are not available.

A Rapid COVID-19 RT-PCR Detection Assay for Low Resource Settings

SARS-CoV-2 is responsible for a highly contagious infection, known as COVID-19. SARS-CoV-2 was discovered in late December 2019 and, since then, has become a global pandemic. Timely and accurate COVID-19 laboratory testing is an essential step in the management of the COVID-19 outbreak. To date, assays based on the reverse-transcription polymerase chain reaction (RT-PCR) in respiratory samples are the gold standard for COVID-19 diagnosis. Unfortunately, RT-PCR has several practical limitations. Consequently, alternative diagnostic methods are urgently required, both for alleviating the pressure on laboratories and healthcare facilities and for expanding testing capacity to enable large-scale screening and ensure a timely therapeutic intervention. To date, few studies have been conducted concerning the potential utilization of rapid testing for COVID-19, with some conflicting results. Therefore, the present systematic review and meta-analysis was undertaken to explore the feasibility of rapid diagnostic tests in the management of the COVID-19 outbreak. Based on ten studies, we computed a pooled sensitivity of 64.8% (95%CI 54.5–74.0), and specificity of 98.0% (95%CI 95.8–99.0), with high heterogeneity and risk of reporting bias. We can conclude that: (1) rapid diagnostic tests for COVID-19 are necessary, but should be adequately sensitive and specific; (2) few studies have been carried out to date; (3) the studies included are characterized by low numbers and low sample power, and (4) in light of these results, the use of available tests is currently questionable for clinical purposes and cannot substitute other more reliable molecular tests, such as assays based on RT-PCR.

Evaluation of eleven rapid tests for detection of antibodies against SARS-CoV-2

Abstract Rapid, scalable, point-of-need, COVID-19 diagnostic testing is necessary to safely re-open economies and prevent future outbreaks. We developed an assay that detects single copies of SARS-CoV-2 virus directly from saliva and swab samples in 30 min using a simple, one-step protocol that utilizes only a heat block and microcentrifuge tube prefilled with a mixture containing the necessary reagents and has a sensitivity and specificity of 97% and 100%, respectively.

Field-deployable, rapid diagnostic testing of saliva samples for SARS-CoV-2.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its associated Coronavirus disease 2019 (COVID-19) pandemic has demanded rapid upscaling of in-vitro diagnostic assays to enable mass screening and testing of high-risk groups, and simultaneous ascertainment of robust data on past SARS-CoV-2 exposure at an individual and population level. To meet the exponential demand in testing, there has been an accelerated development of both molecular and serological assays across a plethora of platforms. In the present review, we discuss the current literature on these modalities including the nucleic acid amplification tests, direct viral antigen tests and the rapidly expanding laboratory based and point of care serological tests. This suite of complementary tests will inform crucial decisions by healthcare providers and policy makers and understanding their strengths and limitations will be critical to their judicious application for the development of algorithmic approaches to treatment and public health strategies.

Testing for SARS-CoV-2 (COVID-19): a systematic review and clinical guide to molecular and serological in-vitro diagnostic assays

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its associated coronavirus disease 2019 (COVID-19) pandemic has demanded rapid upscaling of in-vitro diagnostic assays to enable mass screening and testing of high-risk groups, and simultaneous ascertainment of robust data on past SARS-CoV-2 exposure at an individual and a population level. To meet the exponential demand in testing, there has been an accelerated development of both molecular and serological assays across a plethora of platforms. The present review discusses the current literature on these modalities, including nucleic acid amplification tests, direct viral antigen tests and the rapidly expanding laboratory-based and point of care serological tests. This suite of complementary tests will inform crucial decisions by healthcare providers and policy makers, and understanding their strengths and limitations will be critical to their judicious application for the development of algorithmic approaches to treatment and public health strategies.

On March 11, 2020 the World Health Organization (WHO) upgraded the status of the coronavirus disease 2019 (COVID-19) outbreak from epidemic to a global pandemic. This infection is caused by a novel coronavirus, SARS-CoV-2. Several rapid diagnostic tests have been developed at an astonishing pace; however, COVID-19 requires more highly specific rapid point-of-care diagnostic tests. This review describes the currently available testing approaches, as well as the available test assays including the Xpert® Xpress SARS-CoV-2 test (takes (~)45 min) and Abbott ID COVID-19 test (5 min) as easy to use point-of-care tests for diagnosis of novel COVID-19 that have so far received the US Food and Drug Administration emergency use authorizations clearance. This review is correct as of the date published and will be updated as more diagnostic tests come to light.

Current and emerging diagnostic tests available for the novel COVID-19 global pandemic

OBJECTIVE: With the current SARS-CoV2 outbreak, countless tests need to be performed on potential symptomatic individuals, contacts and travellers. The gold standard is a quantitative polymerase chain reaction (qPCR)-based system taking several hours to confirm positivity. For effective public health containment measures, this time span is too long. We therefore evaluated a rapid test in a high-prevalence community setting. STUDY DESIGN: Thirty-nine randomly selected individuals at a COVID-19 screening centre were simultaneously tested via qPCR and a rapid test. Ten previously diagnosed individuals with known SARS-CoV-2 infection were also analysed. METHODS: The evaluated rapid test is an IgG/IgM-based test for SARS-CoV-2 with a time to result of 20 min. Two drops of blood are needed for the test performance. RESULTS: Of 49 individuals, 22 tested positive by repeated qPCR. In contrast, the rapid test detected only eight of those positive correctly (sensitivity: 36.4%). Of the 27 qPCR-negative individuals, 24 were detected correctly (specificity: 88.9%). CONCLUSION: Given the low sensitivity, we recommend not to rely on an antibody-based rapid test for public health measures such as community screenings.

Rapid point-of-care testing for SARS-CoV-2 in a community screening setting shows low sensitivity

Abstract Objective With the current SARS-CoV2 outbreak, countless tests need to be performed on potential symptomatic individuals, contacts and travellers. The gold standard is a quantitative polymerase chain reaction (qPCR)–based system taking several hours to confirm positivity. For effective public health containment measures, this time span is too long. We therefore evaluated a rapid test in a high-prevalence community setting. Study design Thirty-nine randomly selected individuals at a COVID-19 screening centre were simultaneously tested via qPCR and a rapid test. Ten previously diagnosed individuals with known SARS-CoV-2 infection were also analysed. Methods The evaluated rapid test is an IgG/IgM–based test for SARS-CoV-2 with a time to result of 20 min. Two drops of blood are needed for the test performance. Results Of 49 individuals, 22 tested positive by repeated qPCR. In contrast, the rapid test detected only eight of those positive correctly (sensitivity: 36.4%). Of the 27 qPCR-negative individuals, 24 were detected correctly (specificity: 88.9%). Conclusion Given the low sensitivity, we recommend not to rely on an antibody-based rapid test for public health measures such as community screenings.

Fast and simple high-throughput testing of COVID 19

The recent pandemic of COVID-19 has involved tens of thousands of patients in numerous countries and the causative virus, SARS COV-2 is highly transmissible. Molecular diagnostic tools are central to containment of the virus and initiating proper clinical care. Rapidity, user-friendliness, and high degree of sensitivity and specificity are desirable features of diagnostic assays for screening purposes. Herein, we present a single step reverse transcriptase LAMP assay (RT-LAMP), which can detect up to 500 viral copies in 30 minutes. We challenged our assay with a large number of clinical samples collected from 47 confirmed cases and 213 negative patients. Our LAMP assay showed a high degree of sensitivity and specificity compared to two commercialized qRT-PCR assay as gold standard. We present a rapid RT-LAMP assay that could extend the capacity of laboratories to process 2.5 more clinical samples relative to qRT-PCR and potentially could be used for high-throughput screening purposes.

Development and validation of a rapid single-step reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) system potentially to be used for reliable and high-throughput screening of COVID-19

The ability to rapidly and accurately identify a patient's COVID-19 status has had significant impact on emergency departments (ED) and health systems globally. Since the identification of SARS-CoV-2 illness in the United States, there has been rapid development in patient testing capacity following initial challenges including sparse availability. This was made possible by increasing availability of diagnostic molecular tests in several formats, from laboratory based traditional, RT-PCR methods to near patient testing rapid point of care PCR tests.

Diagnostic Performance of a Rapid Point of Care Test for SARS-CoV-2 in an Urban ED Setting

An ongoing theme of the COVID-19 pandemic is the need for widespread availability of accurate and eﬃcient diagnostic testing for detection of SARS-CoV-2 and antiviral antibodies in infected individuals. This report describes various assay techniques and tests for COVID-19 diagnosis. Most tests for early detection of SARS-CoV-2 RNA rely on the reverse transcription-polymerase chain reaction, but isothermal nucleic acid ampliﬁcation assays, including transcription-mediated ampliﬁcation and CRISPR-based methodologies, are promising alternatives. Identiﬁcation of individuals who have developed antibodies to the SARS-CoV-2 virus requires serological tests, including enzyme-linked immunosorbent assay (ELISA) and lateral ﬂow immunoassay. This report also provides an overview of current development in COVID-19 diagnostic techniques and products to facilitate future improvement and innovation.

Assay Techniques and Test Development for COVID-19 Diagnosis

As the novel coronavirus severe acute respiratory syndrome coronavirus 2 caused coronavirus disease 2019 cases in the United States, the initial test was developed and performed at the Centers for Disease Control and Prevention. As the number of cases increased, the demand for tests multiplied, leading the Centers for Disease Control and Prevention to use the Emergency Utilization Authorization to allow clinical and commercial laboratories to develop tests to detect the presence of the virus. Many nucleic acid tests based on RT-PCR were developed, each with different techniques, specifications, and turnaround time. As the illnesses turned into a pandemic, testing became more crucial. The test supply became inadequate to meet the need and so it had to be prioritized according to guidance. For surveillance, the need for serologic tests emerged. Here, we review the timeline of test development, the turnaround times, and the various approved tests, and compare them as regards the genes they detect. We concentrate on the point-of-care tests and discuss the basis for new serologic tests. We discuss the testing guidance for prioritization and their application in a hospital setting.

Clinical testing for COVID-19

These speed bumps are some of the reasons why many industry watchers and public health officials are praising the development of a 5 min bedside, or point-of-care, test produced by Abbott Laboratories Approved in late March, the test relies on PCR but uses a different method of copying genetic material, allowing it to be done at a constant temperature and quickly Other companies that use this method, called isothermal amplification, for infectious disease diagnostics have developed specialized enzymes that can copy viral RNA without the temperature cycling used in more traditional PCR It’s not clear exactly how Abbott is employing isothermal amplification, but the company says the test can be done in a small, portable machine that many hospitals already use to diagnose other infectious diseases Abbott says this test and another, more traditional PCR diagnostic it developed can perform up to 5 million COVID-19 tests per month total

Companies are racing to develop COVID-19 tests for the US. Will the tests help?

Background: Rapid and extensive testing of large parts of the population and specific subgroups is crucial for proper management of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and decision-making in times of a pandemic outbreak. However, point-of-care (POC) testing in places such as emergency units, outpatient clinics, airport security points or the entrance of any public building is a major challenge. The need for thermal cycling and nucleic acid isolation hampers the use of standard PCR-based methods for this purpose. Methods: To avoid these obstacles, we tested PCR-independent methods for the detection of SARS-CoV-2 RNA from primary material (nasopharyngeal swabs) including loop-mediated isothermal amplification (LAMP) and specific high-sensitivity enzymatic reporter unlocking (SHERLOCK). Results: Whilst specificity of standard LAMP assays appears to be satisfactory, sensitivity does not reach the current gold-standard quantitative real-time polymerase chain reaction (qPCR) assays yet. We describe a novel multiplexed LAMP approach and validate its sensitivity on primary samples. This approach allows for fast and reliable identification of infected individuals. Primer optimization and multiplexing helps to increase sensitivity significantly. In addition, we directly compare and combine our novel LAMP assays with SHERLOCK. Conclusion: In summary, this approach reveals one-step multiplexed LAMP assays as a prime-option for the development of easy and cheap POC test kits.

Rapid SARS-CoV-2 testing in primary material based on a novel multiplex LAMP assay

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.

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

OBJECTIVES: We aimed to evaluate the role of rapid serological tests in the management of coronavirus disease 2019 (COVID-19) patients. METHODS: This retrospective study enrolled 16 real-time reverse transcription polymerase chain reaction-confirmed symptomatic patients with COVID-19 and 58 COVID-19 negative patients at a medical center in Taiwan over a 3-month period. Serial serum samples were collected and tested for antibody response using four point-of-care (POC) lateral flow immunoassays (LFIA) (ALLTEST 2019-nCoV IgG/IgM Rapid Test, Dynamiker 2019-nCoV IgG/IgM Rapid Test, ASK COVID-19 IgG/IgM Rapid Test, and Wondfo SARS-CoV-2 Antibody Test). Time-dependent detection sensitivity and timeliness of seroconversion were determined and compared between the four POC rapid tests. RESULTS: The overall sensitivity and specificity of the four tests for detecting anti-SARS-CoV-2 antibodies after 3 weeks of symptom onset were 100% and 100%, respectively. There was no significant difference between the rapid tests used for detection of IgM and IgG separately and those used for detection of combined total antibody (mainly IgM/IgG). There was no significant difference between the four POC rapid tests in terms of time required for determining seroconversion of COVID-19. Patients with COVID-19 with pneumonia demonstrated shorter seroconversion time than those without pneumonia. CONCLUSION: Though the POC antibody rapid tests based on LFIA showed reliable performance in the detection of SARS-CoV-2-specific antibodies, the results of these tests should be interpreted and applied appropriately in the context of antibody dynamic of COVID-19 infection. COVID-19 patients complicated with pneumonia exhibited earlier anti-SARS-CoV-2 antibody response than COVID-19 patients without pneumonia.

Four point-of-care lateral flow immunoassays for diagnosis of COVID-19 and for assessing dynamics of antibody responses to SARS-CoV-2

The 2019 novel coronavirus (COVID-19) is a newly emerged strain that has never been found in humans before. At present, the laboratory-based reverse transcription-polymerase chain reaction (RT-PCR) is the main method to confirm COVID-19 infection. The intensification of the COVID-19 epidemic overwhelms limited clinical resources in particular, but not only, in developing countries, resulting in many patients not being tested for the infection and in large queues of potentially infected individuals waiting to be tested while providing a breeding ground for the disease. We describe here a rapid, highly sensitive, point-of-care, molecular test amenable for use at home, in the clinic, and at points of entry by minimally trained individuals and with minimal instrumentation. Our test is based on loop mediated isothermal amplification (COVID-19 LAMP) and for higher sensitivity on nested nucleic acid, two stage isothermal amplification (COVID-19 Penn-RAMP). Both tests can be carried out in closed tubes with either fluorescence or colorimetric (e.g., leuco crystal violet LCV) detection. COVID-19 LAMP performs on par with COVID-19 RT-PCR. COVID-19 RAMP has 10 fold better sensitivity than COVID-19 LAMP and COVID-19 RT-PCR when testing purified targets and 100 times better sensitivity than COVID-19 LAMP and COVID-19 RT-PCR when testing rapidly prepared sample mimics. Due to fortunate scarcity of COVID-19 infections in the USA, we were not able to test our assays and methods with patient samples. We hope that such tests will be carried out by colleagues in impacted countries. Our Closed-Tube Penn-RAMP has the potential to significantly reduce false negatives while being amenable to use with minimal instrumentation and training.

A Single and Two-Stage, Closed-Tube, Molecular Test for the 2019 Novel Coronavirus (COVID-19) at Home, Clinic, and Points of Entry.

There is a need for widespread testing in India to stop the spread of the novel coronavirus in the population. While RT-PCR is the recommended diagnostic technique, its use is limited to well-equipped laboratories due to the need for specialized instrumentation, reagents and trained personnel. Immunodiagnostic tests are not yet recommended by the WHO for diagnosing active infections. There is a strong need for developing point-of-care molecular tests. Based on our past experience with paperfluidic devices for diagnosing bacterial infections by molecular tests, we propose the development of a diagnostic test for COVID-19. As a platform technology, it could be adapted to other viral outbreaks in future.

Developing a Point-of-Care Molecular Test to Detect SARS-CoV-2

The emergence of the novel coronavirus SARS-CoV-2 has led to a pandemic infecting more than two million people worldwide in less than four months, posing a major threat to healthcare systems. This is compounded by the shortage of available tests causing numerous healthcare workers to unnecessarily self-isolate. We provide a roadmap instructing how a research institute can be repurposed in the midst of this crisis, in collaboration with partner hospitals and an established diagnostic laboratory, harnessing existing expertise in virus handling, robotics, PCR, and data science to derive a rapid, high throughput diagnostic testing pipeline for detecting SARS-CoV-2 in patients with suspected COVID-19. The pipeline is used to detect SARS-CoV-2 from combined nose-throat swabs and endotracheal secretions/ bronchoalveolar lavage fluid. Notably, it relies on a series of in-house buffers for virus inactivation and the extraction of viral RNA, thereby reducing the dependency on commercial suppliers at times of global shortage. We use a commercial RT-PCR assay, from BGI, and results are reported with a bespoke online web application that integrates with the healthcare digital system. This strategy facilitates the remote reporting of thousands of samples a day with a turnaround time of under 24 hours, universally applicable to laboratories worldwide.

Scalable and Resilient SARS-CoV2 testing in an Academic Centre

COVID-19 testing as sufficient as needed is essential for healthcare workers, patients, and authorities to make informed decisions to confront and eventually defeat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Currently, diagnosis of COVID-19 relies on quantitative reverse-transcription PCR, which is low-throughput, laborious, and often false-negative, making it overwhelmingly challenging to meet testing needs even in industrialized countries. Here we propose a new strategy, which employs a modified loop-mediated isothermal amplification (LAMP) assay, a simple procedure requiring no sophisticated instruments, to index and amplify viral genes from individual specimens, of which the products are readily available for construction of multiplexed libraries for next-generation sequencing. Our strategy would allow precise diagnosis of thousands of specimens in 1-2 days with significantly lower operating expenses. Furthermore, this strategy will make it possible for patients to collect, process, and mail their own samples to facilities for a quick, reliable diagnosis at a population scale.

A high-throughput strategy for COVID-19 testing based on next-generation sequencing

The recent outbreak of the Coronavirus disease 2019 (COVID-19) has quickly spread worldwide since its discovery in Wuhan city, China in December 2019. A comprehensive strategy, including surveillance, diagnostics, research, clinical treatment, and development of vaccines, is urgently needed to win the battle against COVID-19. The past three unprecedented outbreaks of emerging human coronavirus infections at the beginning of the 21st century have highlighted the importance of readily available, accurate, and rapid diagnostic technologies to contain emerging and re-emerging pandemics. Real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) based assays performed on respiratory specimens remain the gold standard for COVID-19 diagnostics. However, point-of-care technologies and serologic immunoassays are rapidly emerging with high sensitivity and specificity as well. Even though excellent techniques are available for the diagnosis of symptomatic patients with COVID-19 in well-equipped laboratories; critical gaps still remain in screening asymptomatic people who are in the incubation phase of the virus, as well as in the accurate determination of live viral shedding during convalescence to inform decisions for ending isolation. This review article aims to discuss the currently available laboratory methods and surveillance technologies available for the detection of COVID-19, their performance characteristics and highlight the gaps in current diagnostic capacity, and finally, propose potential solutions. We also summarize the specifications of the majority of the available commercial kits (PCR, EIA, and POC) for laboratory diagnosis of COVID-19.

Challenges in Laboratory Diagnosis of the Novel Coronavirus SARS-CoV-2.

Challenges in Laboratory Diagnosis of the Novel Coronavirus SARS-CoV-2

COVID-19 rapid antibody cassette point of care tests: practical considerations.

Diagnostic testing to identify persons infected with severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infection is central to control the global pandemic of COVID-19 that began in late 2019. In a few countries, the use of diagnostic testing on a massive scale has been a cornerstone of successful containment strategies. In contrast, the United States, hampered by limited testing capacity, has prioritized testing for specific groups of persons. Real-time reverse transcriptase polymerase chain reaction-based assays performed in a laboratory on respiratory specimens are the reference standard for COVID-19 diagnostics. However, point-of-care technologies and serologic immunoassays are rapidly emerging. Although excellent tools exist for the diagnosis of symptomatic patients in well-equipped laboratories, important gaps remain in screening asymptomatic persons in the incubation phase, as well as in the accurate determination of live viral shedding during convalescence to inform decisions to end isolation. Many affluent countries have encountered challenges in test delivery and specimen collection that have inhibited rapid increases in testing capacity. These challenges may be even greater in low-resource settings. Urgent clinical and public health needs currently drive an unprecedented global effort to increase testing capacity for SARS-CoV-2 infection. Here, the authors review the current array of tests for SARS-CoV-2, highlight gaps in current diagnostic capacity, and propose potential solutions.

Diagnostic Testing for Severe Acute Respiratory Syndrome-Related Coronavirus 2: A Narrative Review

BACKGROUND: In January 2020 reports of unidentified severe respiratory illness were described in Wuhan, China. A rapid expansion in cases affecting most countries around the globe led to major changes in the way people live their daily lives. In the United Kingdom, the Department of Health and Social Care directed healthcare providers to establish additional resources to manage the anticipated surge in cases that could overwhelm the health services. A priority area was testing for SARS-CoV-2 RNA and its detection by qualitative RT-PCR. DESIGN: A laboratory workflow twinning research environment with clinical laboratory capabilities was implemented and validated in the University of Birmingham within 4 days of the project initiation. The diagnostic capability was centred on an IVD CE-marked RT-PCR kit and designed to provide surge capacity to the nearby Queen Elizabeth Hospital. The service was initially tasked with testing healthcare workers (HCW) using throat swabs, and subsequently the process investigated the utility of using saliva as an alternative sample type. RESULTS: Between the 8(th) April 2020 and the 30(th) April 2020, the laboratory tested a total of 1282 HCW for SARS-CoV-2 RNA in throat swabs. RNA was detected in 54% of those who reported symptoms compatible with COVID-19, but in only 4% who were asymptomatic. CONCLUSION: This capability was established rapidly and utilised a cold-chain free methodology, applicable to a wide range of settings, and which can provide surge capacity and support to clinical laboratories facing increasing pressure during periods of national crisis.

Rapid implementation and validation of a cold-chain free SARS-CoV-2 diagnostic testing workflow to support surge capacity

The current standard testing method for screening coronavirus disease 2019 (COVID-19) is through reverse real-time PCR assay (rRT-PCR), a common molecular-based assay that requires an average of four to six hours to provide results [...].

Combining Point-of-Care Diagnostics and Internet of Medical Things (IoMT) to Combat the COVID-19 Pandemic

The recently emerged coronavirus disease COVID-19 has now evolved into a global pandemic. Early detection is crucial for its effective control. Nucleic acid testing for viral pathogen and serological testing for host antibodies are playing important roles in current COVID-19 diagnosis. However, while nucleic acid testing is complicated, facility-restricted and time-consuming, antibody testing may result in high rates of false-negative diagnoses, especially during the early stages of viral infection. Thus, a more rapid and reliable test for both early COVID-19 diagnosis and whole-population screening is urgently needed. Here, we developed a novel nanozyme-based chemiluminescence paper assay for rapid and high-sensitive testing of SARS-CoV-2 spike antigen. Our paper test uses a newly established peroxidase-mimic Co-Fe@hemin nanozyme instead of natural HRP that catalytically amplifies the chemiluminescent signal, allowing for target concentrations to be as low as 0.1 ng/ml. Furthermore, our nanozyme-based chemiluminescence test exhibits a linear range that is 32-fold wider compared to ELISA tests. Importantly, testing is completed in less than 16 min, compared to 1-2 h required for ELISA or nucleic acid tests. Critically, signal detection is feasible using a smartphone camera. Ingredients for our test are simple and readily available, rendering overall cost considerably lower than those used in current diagnoses. In conclusion, our novel test provides a high-sensitive, point-of-care testing (POCT) approach for SARS-CoV-2 antigen detection, which should greatly increase current early screening capacities for suspected infections, and considerably lower demand for national healthcare resources.

Ultra-sensitive nanozyme-based chemiluminescence paper test for rapid diagnosis of SARS-CoV-2 infection

Increasing evidence indicates immunity against severe acute respiratory syndrome coronavirus 2 (sars-cov-2) after covid-19, but it remains unclear for how long the protection remains Serology testing seems to have a higher sensitivity than molecular diagnostics from 8 days after onset of symtoms, and should be part of risk assessment and epidemiological studies of COVID-19 The performance of commercial serological point-of-care (POC) lateral flow tests are highly manufacturer-dependant Low sensitivity increases the risk of false negative results and could result in unnecessary quarantine of test persons with developed antibodies Low specificity increases the risk of false positive results and could lead to false assumptions of immunity Carefully selected serological POC tests for sars-cov-2 can be used in large scale testing but should only be used by licensed medical staff able to understand their limitations and interpret the results

Rapid point-of-care serology testing for sars-cov-2/ Snabbtest for covid-19-antikroppar bor utforas av utbildad personal

Coronavirus disease 2019 (COVID-19), the current uncontrolled outbreak of infectious disease, has caused significant challenges throughout the world. A reliable rapid diagnostic test for COVID-19 is demanded worldwide. The real-time reverse transcriptase polymerase chain was one of the most quickly established methods in the novel viral pandemic and was considered as the gold standard for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this report, we illustrate our experience of applying a protocol from the Taiwan CDC and achieving assay optimization in the immediate circumstances to meet the urgent medical and public health needs.

Optimization of the CDC Protocol of Molecular Diagnosis of COVID-19 for Timely Diagnosis

Increasing evidence indicates immunity against severe acute respiratory syndrome coronavirus 2 (sars-cov-2) after covid-19, but it remains unclear for how long the protection remains. Serology testing seems to have a higher sensitivity than molecular diagnostics from 8 days after onset of symtoms, and should be part of risk assessment and epidemiological studies of COVID-19. The performance of commercial serological point-of-care (POC) lateral flow tests are highly manufacturer-dependant. Low sensitivity increases the risk of false negative results and could result in unnecessary quarantine of test persons with developed antibodies. Low specificity increases the risk of false positive results and could lead to false assumptions of immunity. Carefully selected serological POC tests for sars-cov-2 can be used in large scale testing but should only be used by licensed medical staff able to understand their limitations and interpret the results.

[Rapid point-of-care serology testing for sars-cov-2].

An epidemic caused by an outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in China in December 2019 has since rapidly spread internationally, requiring urgent response from the clinical diagnostics community. We present a detailed overview of the clinical validation and implementation of the first laboratory-developed real-time reverse-transcription-PCR (rRT-PCR) test offered in the NewYork-Presbyterian Hospital system following the emergency use authority (EUA) guidance issued by the US Food and Drug Administration. Validation was performed on nasopharyngeal and sputum specimens (n=124) using newly designed dual-target rRT-PCR (altona RealStar SARS-CoV-2 Reagent) for detecting of SARS-CoV-2 in upper respiratory and lower respiratory tract specimens, including bronchoalveolar lavage and tracheal aspirates. Accuracy testing demonstrated excellent assay agreement between expected and observed values. The limit of detection (LOD) was 2.7 and 23.0 gene copies/reaction for nasopharyngeal and sputum specimens, respectively. Retrospective analysis of 1,694 tests from 1,571 patients revealed increased positivity in older patients and males compared to females, and an increasing positivity rate from approximately 20% at the start of testing to 50% at the end of testing three weeks later. Our findings demonstrate that the assay accurately and sensitively identifies SARS-CoV-2 in multiple specimen types in the clinical setting and summarizes clinical data from early in the epidemic in New York City.

Rapid implementation of SARS-CoV-2 emergency use authorization RT-PCR testing and experience at an academic medical institution

The SARS-CoV-2 pandemic has evolved far more aggressively in countries lacking a robust testing strategy to identify infected individuals. Given the global demand for fast and reliable diagnosis to determine the carrier individuals, a stock-out scenario for a number of essential reagents/kits used along the diagnostic process has been foreseen by many organizations. Having identified the RNA extraction step as one of the key bottlenecks, we tested several alternatives that avoid the use of commercial kits for this step. The analysis showed that 2-propanol precipitation of the viral RNA, followed by one-step RT-qPCR results in a sensitivity and specificity comparable to that provided currently by automatized systems such as the COBAS 6800 system. Therefore, this simple protocol allows SARS-CoV-2 testing independently of commercial kit providers in a time and cost-effective manner. It can be readily implemented in research and/or diagnostic laboratories worldwide, provided that patient confidentiality and researcher safety are ensured. Scaling up the testing capabilities of hospitals and research facilities will identify larger numbers of infected individuals to paint a clear picture of the COVID-19 prevalence, a pre-requisite for informed policy decision making.

Fast SARS-CoV-2 detection protocol based on RNA precipitation and RT-qPCR in nasopharyngeal swab samples

In the context of the Covid-19 pandemic, the development and validation of rapid and easy-to-perform diagnostic methods are of high priority. We compared the performance of four rapid antigen detection tests for SARS-CoV-2 in respiratory samples. Immunochromatographic SARS-CoV-2 assays from RapiGEN, Liming bio, Savant, and Bioeasy were evaluated using universal transport medium containing naso-oropharyngeal swabs from suspected Covid-19 cases. The diagnostic accuracy was determined in comparison to SARS-CoV-2 RT-PCR. A total of 111 samples were included; 80 were RT-PCR positive. Median patients’ age was 40 years, 55% were female, and 88% presented within the first week after symptom onset. The evaluation of the Liming bio assay was discontinued due to insufficient performance. The overall sensitivity values of RapiGEN, Liming bio, and Bioeasy tests were 62.0% (CI95% 51.0–71.9), 16.7% (CI95% 10.0–26.5), and 85.0% (CI95% 75.6–91.2), respectively, with specificities of 100%. Sensitivity was significantly higher in samples with high viral loads (RapiGEN, 84.9%; Bioeasy, 100%). The study highlighted the significant heterogeneity of test performance among evaluated assays, which might have been influenced by the use of a non-validated sample material. The high sensitivity of some tests demonstrated that rapid antigen detection has the potential to serve as an alternative diagnostic method, especially in patients presenting with high viral loads in early phases of infection. This is particularly important in situations with limited access to RT-PCR or prolonged turnaround time. Further comparative evaluations are necessary to select products with high performance among the growing market of diagnostic tests for SARS-CoV-2.

Head-to-head comparison of four antigen-based rapid detection tests for the diagnosis of SARS-CoV-2 in respiratory samples

The ongoing novel coronavirus (COVID-19) outbreak as a global public health emergency infected by SARC-CoV-2 has caused devastating loss around the world. Currently, a lot of diagnosis methods have been used to detect the infection. The nucleic acid (NA) testing is reported to be the clinical standard for COVID-19 infection. Evidence shows that a faster and more convenient method to detect in the early phase will control the spreading of SARS-CoV-2. Here, we propose a method to detect SARC-Cov-2 infection within two hours combined with Loop-mediated Isothermal Amplification (LAMP) reaction and nanopore Flongle workflow. In this approach, RNA reverse transcription and nucleic acid amplification reaction with one step in 30 minutes at 60-65°C constant temperature environment, nanopore Flongle rapidly adapter ligated within 10 minutes. Flongle flow cell sequencing and analysis in real-time. This method described here has the advantages of rapid amplification, convenient operation and real-time detection which is the most important for rapid and reliable clinical diagnosis of COVID-19. Moreover, this approach not only can be used for SARS-CoV-2 detection but also can be extended to other respiratory viruses and pathogens.

Rapid detection of SARS-CoV-2 and other respiratory viruses by using LAMP method with Nanopore Flongle workflow

Pooling of samples can increase lab capacity when using Polymerase chain reaction (PCR) to detect infections such as COVID-However, pool testing is typically performed via an adaptive testing strategy which requires a feedback loop in the lab and at least two PCR runs to confirm positive results. This can cost precious time. We discuss a non-adaptive testing method where each sample is distributed in a prescribed manner over several pools, and which yields reliable results after one round of testing. More precisely, assuming knowledge about the overall infection incidence rate, we calculate explicit error bounds on the number of false positives which scale very favourably with pool size and sample multiplicity. This allows for hugely streamlined PCR testing and cuts in detection times for a large-scale testing scenario. A viable consequence of this method could be real-time screening of entire communities, frontline healthcare workers and international flight passengers, for example, using the PCR machines currently in operation.

Rapid, Large-Scale, and Effective Detection of COVID-19 Via Non-Adaptive Testing

As the COVID-19 outbreak has evolved in each country, the approach to the laboratory assessment of SARS-CoV-2 infection has had to evolve as well. This review addresses the evolving approach to the laboratory assessment of COVID-19 and discusses how algorithms for testing have been driven, in part, by the demand for testing overwhelming the capacity to accomplish such testing. This review focused on testing in the United States as this testing is evolving whereas in China and other countries such as South Korea testing is widely available and includes both molecular testing for SARS-CoV-2 as well as serological testing using both ELISA methodology and lateral flow immunoassay methodology. Although commercial testing systems are becoming available, there will likely be insufficient numbers of such tests due to high demand. Serological testing will be the next testing issue as the COVID-19 begins to subside. This will allow immunity testing as well as will allow the parameters of the COVID-19 outbreak to be defined. This article is protected by copyright. All rights reserved.

An Evolving Approach to the Laboratory Assessment of COVID-19

Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold standard diagnostic assays are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment (PPE), instrumentation, and labor. Here we present an approach to overcome these challenges with the development of a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe our optimizations of the LAMP reaction and saliva pre-treatment protocols that enabled rapid and sensitive detection of < 10(2) viral genomes per reaction in contrived saliva controls. We also observed high performance of this assay on a limited number of clinical saliva samples. While thorough validation on additional clinical samples will be needed before such an assay can be widely used, these preliminary results demonstrate a promising approach to overcome the current bottlenecks limiting widespread testing.

Rapid and extraction-free detection of SARS-CoV-2 from saliva with colorimetric LAMP

Following the first reports of coronavirus disease-19 (COVID-19) by China to the World Health Organization (WHO) on 31st December 2019, more than 4,302,774 novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) cases have been reported by authorities in 212 countries and territories by 12th May 2020. The outbreak and spread of COVID-19 worldwide, highlights the critical need for developing rapid and accurate diagnostic testing methods for emerging human coronavirus (CoV) infections. Testing is crucial to track the spread of disease during a pandemic, and to swiftly permit public health interventions including isolation, quarantine, and appropriate clinical management of afflicted individuals. The key components of viral diagnostic tests are (1) collection of the appropriate sample (blood, nasal swab, and throat swab), (2) availability of the genetic and proteomic sequences of the novel virus for analysis, and (3) rapid and accurate laboratory testing methods. The current gold standard for the molecular diagnosis of SARS-CoV-2 infection is the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for the qualitative and quantitative detection of viral nucleic acids. Other relevant laboratory methods include enzyme-linked immunoassays (EIA) for viral antibody and antigen detection, and serum viral neutralization (SVN) assays for antibody neutralization determination. The challenges faced in developing a diagnostic test for a novel pathogen are the ability to measure low viral loads for early detection, to provide low or no cross-reactivity with other viral strains and to deliver results rapidly. Several point-of-care molecular devices are currently being integrated for fast and accurate diagnosis of SARS-CoV-2 infections. This review discusses the current laboratory methods available to test for coronaviruses by focusing on the present COVID-19 outbreak.

Laboratory Testing Methods for Novel Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2)

We present here INSIGHT (Isothermal NASBA-Sequencing based hIGH-througput Test): a two-stage COVID-19 testing strategy, using a combination of an isothermal NASBA reaction and next generation sequencing. From commercially acquired human saliva with spiked-in viral RNA as input, the first stage employs isothermal amplification of viral RNA to give a rapid result in one to two hours, using either fluorescence detection or a dipstick readout, whilst simultaneously incorporating sample-specific barcodes into the amplification product. In the first stage, fluorescent viral RNA detection can be consistently achieved at 10-100 copies per 20 µl reaction. The second stage pools post-amplification barcoded products from multiple samples for scalable sequencing that could be centralised, to further improve the accuracy of the test in a massively parallel way. Our two-stage testing strategy is suitable for further development into a home-based or point-of-care assay, and is potentially scalable to population level. IMPORTANT This protocol has not been validated on patient samples and should not be used for clinical diagnosis without further validation and certification. ***This is ongoing research to develop a testing strategy for the SARS-CoV-2 virus. The Wellcome Sanger Institute is not in a position to develop this into a commercial product, but would be open to discussions with third parties about how they could develop this further to achieve the societal benefits of this work.***

INSIGHT: a scalable isothermal NASBA-based platform for COVID-19 diagnosis

The current coronavirus disease 2019 (COVID-19) pandemic is largely driven by community transmission, after 2019 novel Coronavirus (2019-nCoV or SARS-CoV-2) crosses the borders. To stop the spread, rapid testing is required at community clinics and hospitals. These rapid tests should be comparable with the standard PCR technology. Isothermal amplification technology provides an excellent alternative that is highly amenable to resource limited settings, where expertise and infrastructure to support PCR are not available. In this review, we provide a brief description of isothermal amplification technology, its potential and the gaps that need to be considered for SARS-CoV-2 detection. Among this emerging technology, loop-mediated amplification (LAMP), recombinase polymerase amplification (RPA) and Nicking enzyme-assisted reaction (NEAR) technologies have been identified as potential platforms that could be implemented at community level, without samples referral to a centralized laboratory and prolonged turnaround time associated with the standard COVID-19 RT-PCR test. LAMP, for example, has recently been shown to be comparable with PCR and could be performed in less than 30 min by non-laboratory staff, without RNA extractions commonly associated with PCR. Interestingly, NEAR (ID NOW™ COVID-19 (Abbott, IL, USA) was able to detect the virus in 5 min. More so, isothermal platforms are cost effective and could easily be scaled up to resource limited settings. Diagnostics developers, scientific community and commercial companies could consider this alternative method to help stop the spread of COVID-19.

COVID-19 Infection Diagnosis: Potential Impact of Isothermal Amplification Technology to Reduce Community Transmission of SARS-CoV-2

The world needs mass at-home serological testing for antibodies elicited by SARS-CoV-2, and rapid and frequent point-of-care testing for the presence of the virus’ RNA in selected populations.

Humanity tested

In the last few months, an unprecedented number of laboratory tests for COVID‐19 have been developed at a remarkable speed. With the rapid adoption of these tests into clinical practice, combined with the widespread publicity they received, questions arose related to the different types of tests, their utility, performance, and regulatory approval status. The aim of this publication is to provide a general landscape of laboratory testing for COVID‐19 and offer a historical and regulatory perspective associated with them. Specifically, we aim to elaborate on the regulatory complexities of diagnostic testing in the U.S. and its implications to the present outbreak, as well as provide a synopsis of laboratory tests that have been developed for COVID‐19. We will first address the detection of Sars‐Cov‐2 directly by either nucleic acid amplification tests (NAAT) or by the detection of the viral protein for active infections. Subsequently, we will provide an overview of serological tests that can aid not only in diagnosis but additionally help to identify prior infections and potential immunity.

Testing for SARS‐CoV‐2: the day the world turned its attention to the clinical laboratory

BACKGROUND: In January 2020 reports of unidentified severe respiratory illness were described in Wuhan, China. A rapid expansion in cases affecting most countries around the globe led to major changes in the way people live their daily lives. In the United Kingdom, the Department of Health and Social Care directed healthcare providers to establish additional resources to manage the anticipated surge in cases that could overwhelm the health services. A priority area was testing for SARS-CoV-2 RNA and its detection by qualitative RT-PCR. DESIGN: A laboratory workflow twinning research environment with clinical laboratory capabilities was implemented and validated in the University of Birmingham within 4 days of the project initiation. The diagnostic capability was centred on an IVD CE-marked RT-PCR kit and designed to provide surge capacity to the nearby Queen Elizabeth Hospital. The service was initially tasked with testing healthcare workers (HCW) using throat swabs, and subsequently the process investigated the utility of using saliva as an alternative sample type. RESULTS: Between the 8th April 2020 and the 30th April 2020, the laboratory tested a total of 1282 HCW for SARS-CoV-2 RNA in throat swabs. RNA was detected in 54 % of those who reported symptoms compatible with COVID-19, but in only 4% who were asymptomatic. CONCLUSION: This capability was established rapidly and utilised a cold-chain free methodology, applicable to a wide range of settings, and which can provide surge capacity and support to clinical laboratories facing increasing pressure during periods of national crisis.

Rapid implementation and validation of a cold-chain free SARS-CoV-2 diagnostic testing workflow to support surge capacity

In vitro diagnostics of coronavirus disease 2019: Technologies and application

The recent outbreak of the novel coronavirus SARS-CoV-2, which causes COVID-19, can be diagnosed using RT-qPCR, but inadequate access to reagents and equipment has slowed disease detection and impeded efforts to mitigate viral spread. Alternative approaches based on combinations of isothermal amplification and CRISPR-mediated detection, such as the SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) technique, offer reduced dependence on RT-qPCR equipment, but previously reported methods required multiple fluid handling steps, complicating their deployment outside clinical labs. Here we developed a simple test chemistry called STOP (SHERLOCK Testing in One Pot) for detecting SARS-CoV-2 in one hour that is suitable for point-of-care use. This simplified test, STOPCovid, provides sensitivity comparable to RT-qPCR-based SARS-CoV-2 tests and has a limit of detection of 100 copies of viral genome input in saliva or nasopharyngeal swabs per reaction. Using lateral flow readout, the test returns result in 70 minutes, and using fluorescence readout, the test returns result in 40 minutes. Moreover, we validated STOPCovid using nasopharyngeal swabs from COVID-19 patients and were able to correctly diagnose 12 positive and 5 negative patients out of 3 replicates. We envision that implementation of STOPCovid will significantly aid “test-trace-isolate” efforts, especially in low-resource settings, which will be critical for long-term public health safety and effective reopening of the society.

Point-of-care testing for COVID-19 using SHERLOCK diagnostics

The previous outbreaks of SARS-CoV and MERS-CoV have led researchers to study the role of diagnostics in impediment of further spread and transmission. With the recent emergence of the novel SARS-CoV-2, the availability of rapid, sensitive, and reliable diagnostic methods is essential for disease control. Hence, we have developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the specific detection of SARS-CoV-2. The primer sets for RT-LAMP assay were designed to target the nucleocapsid gene of the viral RNA, and displayed a detection limit of 102 RNA copies close to that of qRT-PCR. Notably, the assay has exhibited a rapid detection span of 30â min combined with the colorimetric visualization. This test can detect specifically viral RNAs of the SARS-CoV-2 with no cross-reactivity to related coronaviruses, such as HCoV-229E, HCoV-NL63, HCoV-OC43, and MERS-CoV as well as human infectious influenza viruses (type B, H1N1pdm, H3N2, H5N1, H5N6, H5N8, and H7N9), and other respiratory disease-causing viruses (RSVA, RSVB, ADV, PIV, MPV, and HRV). Furthermore, the developed RT-LAMP assay has been evaluated using specimens collected from COVID-19 patients that exhibited high agreement to the qRT-PCR. Our RT-LAMP assay is simple to perform, less expensive, time-efficient, and can be used in clinical laboratories for preliminary detection of SARS-CoV-2 in suspected patients. In addition to the high sensitivity and specificity, this isothermal amplification conjugated with a single-tube colorimetric detection method may contribute to the public health responses and disease control, especially in the areas with limited laboratory capacities.

Development of a reverse transcription-loop-mediated isothermal amplification as a rapid early-detection method for novel SARS-CoV-2

Diagnostic testing to identify persons infected with severe acute respiratory syndrome–related coronavirus-2 (SARS–CoV-2) infection is central to control the global pandemic of COVID-19 that began in late 2019. In a few countries, the use of diagnostic testing on a massive scale has been a cornerstone of successful containment strategies. In contrast, the United States, hampered by limited testing capacity, has prioritized testing for specific groups of persons. Real-time reverse transcriptase polymerase chain reaction–based assays performed in a laboratory on respiratory specimens are the reference standard for COVID-19 diagnostics. However, point-of-care technologies and serologic immunoassays are rapidly emerging. Although excellent tools exist for the diagnosis of symptomatic patients in well-equipped laboratories, important gaps remain in screening asymptomatic persons in the incubation phase, as well as in the accurate determination of live viral shedding during convalescence to inform decisions to end isolation. Many affluent countries have encountered challenges in test delivery and specimen collection that have inhibited rapid increases in testing capacity. These challenges may be even greater in low-resource settings. Urgent clinical and public health needs currently drive an unprecedented global effort to increase testing capacity for SARS–CoV-2 infection. Here, the authors review the current array of tests for SARS–CoV-2, highlight gaps in current diagnostic capacity, and propose potential solutions.

Diagnostic Testing for Severe Acute Respiratory Syndrome–Related Coronavirus-2: A Narrative Review

The previous outbreaks of SARS-CoV and MERS-CoV have led researchers to study the role of diagnostics in impediment of further spread and transmission. With the recent emergence of the novel SARS-CoV-2, the availability of rapid, sensitive, and reliable diagnostic methods is essential for disease control. Hence, we have developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the specific detection of SARS-CoV-2. The primer sets for RT-LAMP assay were designed to target the nucleocapsid gene of the viral RNA, and displayed a detection limit of 10(2) RNA copies close to that of qRT-PCR. Notably, the assay has exhibited a rapid detection span of 30 min combined with the colorimetric visualization. This test can detect specifically viral RNAs of the SARS-CoV-2 with no cross-reactivity to related coronaviruses, such as HCoV-229E, HCoV-NL63, HCoV-OC43, and MERS-CoV as well as human infectious influenza viruses (type B, H1N1pdm, H3N2, H5N1, H5N6, H5N8, and H7N9), and other respiratory disease-causing viruses (RSVA, RSVB, ADV, PIV, MPV, and HRV). Furthermore, the developed RT-LAMP assay has been evaluated using specimens collected from COVID-19 patients that exhibited high agreement to the qRT-PCR. Our RT-LAMP assay is simple to perform, less expensive, time-efficient, and can be used in clinical laboratories for preliminary detection of SARS-CoV-2 in suspected patients. In addition to the high sensitivity and specificity, this isothermal amplification conjugated with a single-tube colorimetric detection method may contribute to the public health responses and disease control, especially in the areas with limited laboratory capacities.

The recent outbreak of a novel coronavirus SARS-CoV-2 (also known as 2019-nCoV) threatens global health, given serious cause for concern. SARS-CoV-2 is a human-to-human pathogen that caused fever, severe respiratory disease and pneumonia (known as COVID-19). By press time, more than 70,000 infected people had been confirmed worldwide. SARS-CoV-2 is very similar to the severe acute respiratory syndrome (SARS) coronavirus broke out 17 years ago. However, it has increased transmissibility as compared with the SARS-CoV, e.g. very often infected individuals without any symptoms could still transfer the virus to others. It is thus urgent to develop a rapid, accurate and onsite diagnosis methods in order to effectively identify these early infects, treat them on time and control the disease spreading. Here we developed an isothermal LAMP based method-iLACO (isothermal LAMP based method for COVID-19) to amplify a fragment of the ORF1ab gene using 6 primers. We assured the species-specificity of iLACO by comparing the sequences of 11 related viruses by BLAST (including 7 similar coronaviruses, 2 influenza viruses and 2 normal coronaviruses). The sensitivity is comparable to Taqman based qPCR detection method, detecting synthesized RNA equivalent to 10 copies of 2019-nCoV virus. Reaction time varied from 15-40 minutes, depending on the loading of virus in the collected samples. The accuracy, simplicity and versatility of the new developed method suggests that iLACO assays can be conveniently applied with for 2019-nCoV threat control, even in those cases where specialized molecular biology equipment is not available.

Rapid colorimetric detection of COVID-19 coronavirus using a reverse tran-scriptional loop-mediated isothermal amplification (RT-LAMP) diagnostic plat-form: iLACO

OBJECTIVES: To assess the diagnostic performance of rapid lateral flow immunochromatographic assays (LFAs) compared to an enzyme-linked immunosorbent assay (ELISA) and nucleic acid amplification tests (NATs) in suspected coronavirus disease 2019 (COVID-19) patients. METHODS: Patients presenting to a Dutch teaching hospital were eligible between March 17 and April 10, 2020, when they had respiratory symptoms that were suspected for COVID-19. The performances of six different LFAs were evaluated in plasma samples obtained on corresponding respiratory sample dates of NATs testing. Subsequently, the best performing LFA was evaluated in 228 patients and in 50 sera of a historical patient control group. RESULTS: In the pilot analysis sensitivity characteristics of LFA were heterogenous ranging from 2/20 (10%; 95% confidence interval (CI) 0-23) to 11/20 (55%; 95% CI 33-77). In the total cohort, Orient Gene Biotech COVID-19 IgG/IgM Rapid Test LFA had a sensitivity of 43/99 (43%; 95% CI 34-53) and specificity of 126/129 (98%; 95% CI 95-100). Sensitivity increased to 31/52 (60%; 95% CI 46-73) in patients with at least seven days of symptoms, and to 21/33 (64%; 95% CI 47-80) in patients with C-reactive protein (CRP) >100 mg/L. Sensitivity and specificity of Wantai SARS-CoV-2 Ab ELISA was 59/95 (62%; 95% CI 52-72) and 125/128 (98%; 95% CI 95-100) in all patients, respectively, but sensitivity increased to 38/48 (79%; 95% CI 68-91) in patients with at least seven days of symptoms. CONCLUSIONS: There is large variability in diagnostic test performance between rapid LFAs, but overall limited sensitivity and high specificity in acutely admitted patients. Sensitivity improved in patients with longer existing symptoms or high CRP. LFAs should only be considered as additional triage tools when these may lead to the improvement of hospital logistics.

Comparison of diagnostic accuracies of rapid serological tests and ELISA to molecular diagnostics in patients with suspected COVID-19 presenting to the hospital

OBJECTIVES: To assess the diagnostic performance of rapid lateral flow immunochromatographic assays (LFAs) compared with an ELISA and nucleic acid amplification tests (NATs) in individuals with suspected coronavirus disease 2019 (COVID-19). METHODS: Patients presenting to a Dutch teaching hospital were eligible between 17 March and 10 April 2020, when they had respiratory symptoms that were suspected for COVID-19. The performances of six different LFAs were evaluated in plasma samples obtained on corresponding respiratory sample dates of NATs testing. Subsequently, the best performing LFA was evaluated in 228 patients and in 50 sera of a historical patient control group. RESULTS: In the pilot analysis, sensitivity characteristics of LFA were heterogeneous, ranging from 2/20 (10%; 95% CI 0%-23%) to 11/20 (55%; 95% CI 33%-77%). In the total cohort, Orient Gene Biotech COVID-19 IgG/IgM Rapid Test LFA had a sensitivity of 43/99 (43%; 95% CI 34%-53%) and specificity of 126/129 (98%; 95% CI 95%-100%). Sensitivity increased to 31/52 (60%; 95% CI 46%-73%) in patients with at least 7 days of symptoms, and to 21/33 (64%; 95% CI 47%-80%) in patients with C-reactive protein (CRP) ≥100 mg/L. Sensitivity and specificity of Wantai SARS-CoV-2 Ab ELISA was 59/95 (62%; 95% CI 52%-72%) and 125/128 (98%; 95% CI 95%-100%) in all patients, respectively, but sensitivity increased to 38/48 (79%; 95% CI 68%-91%) in patients with at least 7 days of symptoms. CONCLUSIONS: There is large variability in diagnostic test performance between rapid LFAs, but overall limited sensitivity and high specificity in acutely admitted patients. Sensitivity improved in patients with longer existing symptoms or high CRP. LFAs should only be considered as additional triage tools when these may lead to the improvement of hospital logistics.

Comparison of diagnostic accuracies of rapid serological tests and ELISA to molecular diagnostics in patients with suspected coronavirus disease 2019 presenting to the hospital

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the need for different types of diagnostics, comparative validation of new tests, faster approval by federal agencies, and rapid production of test kits to meet global demands. In this Perspective, we discuss the utility and challenges of current diagnostics for COVID-19.

COVID-19 diagnostics in context.

COVID-19 pandemic is a serious global health issue today due to the rapid human to human transmission of SARS-CoV-2, a new type of coronavirus that causes fatal pneumonia. SARS -CoV-2 has a faster rate of transmission than other coronaviruses such as SARS and MERS and until now there are no approved specific drugs or vaccines for treatment. Thus, early diagnosis is crucial to prevent the extensive spread of the disease. The reverse transcription-polymerase chain reaction (RT-PCR) is the most routinely used method until now to detect SARS-CoV-2 infections. However, several other faster and accurate assays are being developed for the diagnosis of COVID-19 aiming to control the spread of infection through the identification of patients and immediate isolation. In this review, we will discuss the various detection methods of the SARS-CoV-2 virus including the recent developments in immunological assays, amplification techniques as well as biosensors.

Diagnostic techniques for COVID-19 and new developments

The current COVID-19 pandemic presents a serious public health crisis, and a better understanding of the scope and spread of the virus would be aided by more widespread testing. Nucleic-acid based tests currently offer the most sensitive and early detection of COVID-19. However, the "gold standard" test pioneered by the United States Center for Disease Control & Prevention, takes several hours to complete and requires extensive human labor, materials such as RNA extraction kits that could become in short supply and relatively scarce qPCR machines. It is clear that a huge effort needs to be made to scale up current COVID-19 testing by orders of magnitude. There is thus a pressing need to evaluate alternative protocols, reagents, and approaches to allow nucleic-acid testing to continue in the face of these potential shortages. There has been a tremendous explosion in the number of papers written within the first weeks of the pandemic evaluating potential advances, comparable reagents, and alternatives to the "gold-standard" CDC RT-PCR test. Here we present a collection of these recent advances in COVID-19 nucleic acid testing, including both peer-reviewed and preprint articles. Due to the rapid developments during this crisis, we have included as many publications as possible, but many of the cited sources have not yet been peer-reviewed, so we urge researchers to further validate results in their own labs. We hope that this review can urgently consolidate and disseminate information to aid researchers in designing and implementing optimized COVID-19 testing protocols to increase the availability, accuracy, and speed of widespread COVID-19 testing.

Overcoming the bottleneck to widespread testing: A rapid review of nucleic acid testing approaches for COVID-19 detection.

The current COVID-19 pandemic presents a serious public health crisis, and a better understanding of the scope and spread of the virus would be aided by more widespread testing. Nucleic-acid-based tests currently offer the most sensitive and early detection of COVID-19. However, the "gold standard" test pioneered by the U.S. Centers for Disease Control and Prevention takes several hours to complete and requires extensive human labor, materials such as RNA extraction kits that could become in short supply, and relatively scarce qPCR machines. It is clear that a huge effort needs to be made to scale up current COVID-19 testing by orders of magnitude. There is thus a pressing need to evaluate alternative protocols, reagents, and approaches to allow nucleic-acid testing to continue in the face of these potential shortages. There has been a tremendous explosion in the number of papers written within the first weeks of the pandemic evaluating potential advances, comparable reagents, and alternatives to the "gold-standard" CDC RT-PCR test. Here we present a collection of these recent advances in COVID-19 nucleic acid testing, including both peer-reviewed and preprint articles. Due to the rapid developments during this crisis, we have included as many publications as possible, but many of the cited sources have not yet been peer-reviewed, so we urge researchers to further validate results in their own laboratories. We hope that this review can urgently consolidate and disseminate information to aid researchers in designing and implementing optimized COVID-19 testing protocols to increase the availability, accuracy, and speed of widespread COVID-19 testing.

Overcoming the bottleneck to widespread testing: a rapid review of nucleic acid testing approaches for COVID-19 detection

The COVID-19 pandemic provides an urgent example where a gap exists between availability of state-of-the-art diagnostics and current needs. As assay details and primer sequences become widely known, many laboratories could perform diagnostic tests using methods such as RT-PCR or isothermal RT-LAMP amplification. A key advantage of RT-LAMP based approaches compared to RT-PCR is that RT-LAMP is known to be robust in detecting targets from unprocessed samples. In addition, RT-LAMP assays are performed at a constant temperature enabling speed, simplicity, and point-of-use testing. Here, we provide the details of an RT-LAMP isothermal assay for the detection of SARS-CoV-2 virus with performance comparable to currently approved tests using RT-PCR. We characterize the assay by introducing swabs in virus spiked synthetic nasal fluids, moving the swab to viral transport medium (VTM), and using a volume of that VTM for performing the amplification without an RNA extraction kit. The assay has a Limit-of-Detection (LOD) of 50 RNA copies/μL in the VTM solution within 20 minutes, and LOD of 5000 RNA copies/μL in the nasal solution. Additionally, we show the utility of this assay for real-time point-of-use testing by demonstrating detection of SARS-CoV-2 virus in less than 40 minutes using an additively manufactured cartridge and a smartphone-based reader. Finally, we explore the speed and cost advantages by comparing the required resources and workflows with RT-PCR. This work could accelerate the development and availability of SARS-CoV-2 diagnostics by proving alternatives to conventional laboratory benchtop tests.

Rapid Isothermal Amplification and Portable Detection System for SARS-CoV-2

The pandemic of novel coronavirus disease 2019 (COVID-19) seriously threatened the public health all over the world. A colloidal gold immunochromatography assay for IgM/IgG antibodies against the receptor-binding domain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S1 protein was established to assess its rapid diagnostic value. We first designed and manufactured all contents of the test cassette of SARS-CoV-2 rapid test kit: the colloidal gold-labeled mouse-antihuman lgM/lgG antibody, the recombinant SARS-CoV-2 antigen, the nitrocellulose membrane control line, and specimen diluents. Furthermore, reverse transcription-polymerase chain reaction (RT-PCR) assay, colloidal gold immunochromatography assay, serological validation of cross reaction with other common viruses, and clinical validation were performed. The kit was finally evaluated by 75 serum/plasma samples of SARS-CoV-2 infection cases and 139 healthy samples as control, with the result of that the sensitivity, specificity, and accuracy for IgM were 90.67%, 97.84%, and 95.33%, whereas for IgG were 69.33%, 99.28%, and 88.79%, respectively; the combination of IgM and IgG could improve the value: 92.00%, 97.12%, and 95.33%, respectively. Therefore, the rapid detection kit has high sensitivity and specificity, especially for IgM&IgG, showing a critical value in clinical application and epidemic control of COVID-19.

A new and rapid approach for detecting COVID-19 based on S1 protein fragments.

Background: The need for a fast and reliable test for COVID-19 is paramount in managing the current pandemic. A cost effective and efficient diagnostic tool as near to the point of care (PoC) as possible would be a game changer in current testing. We tested reverse transcription loop mediated isothermal amplification (RT-LAMP), a method which can produce results in under 30 minutes, alongside standard methods in a real-life clinical setting. Methods: This service improvement project piloted a research RT-LAMP method on nasal and pharyngeal swabs on 21 residents in an NHS Category 1 care home, with two index COVID-19 cases, and compared it to multiplex tandem reverse transcription polymerase chain reaction (RT-PCR). We calculated the sensitivity, specificity, positive and negative predictive values of a single RT-LAMP swab compared to RT-PCR, as per STARD guidelines. We also recorded vital signs of patients to correlate clinical and laboratory information. Findings: The novel method accurately detected 8/10 PCR positive cases and identified a further 3 positive cases. Eight further cases were negative using both methods. Using repeated RT-PCR as a 'gold standard', the sensitivity and specificity of the novel test were 80% and 73% respectively. Positive predictive value (PPV) was 73% and negative predictive value (NPV) was 83%. We also observed hypothermia to be a significant early clinical sign in a number of COVID-19 patients in this setting. Interpretation: RT-LAMP testing for SARS-CoV-2 was found to be promising, fast, easy to use and to work equivalently to RT-PCR methods. Definitive studies to evaluate this method in larger cohorts are underway. RT-LAMP has the potential to transform COVID-19 detection, bringing rapid and accurate testing to the point of care. This method could be deployed in mobile testing units in the community, care homes and hospitals to detect disease early and prevent spread.

Detecting SARS-CoV-2 at point of care: Preliminary data comparing Loop-mediated isothermal amplification (LAMP) to PCR

There is an ongoing worldwide coronavirus disease 2019 (Covid-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At present, confirmatory diagnosis is by reverse transcription polymerase chain reaction (RT-PCR), typically taking several hours and requiring a molecular laboratory to perform. There is an urgent need for rapid, simplified, and cost-effective detection methods. We have developed and analytically validated a protocol for direct rapid extraction-free PCR (DIRECT-PCR) detection of SARS-CoV-2 without the need for nucleic acid purification. As few as six RNA copies per reaction of viral nucleocapsid (N) gene from respiratory samples such as sputum and nasal exudate can be detected directly using our one-step inhibitor-resistant assay. The performance of this assay was validated on a commercially available portable PCR thermocycler. Viral lysis, reverse transcription, amplification, and detection are achieved in a single-tube homogeneous reaction within 36 min. This minimizes hands-on time, reduces turnaround-time for sample-to-result, and obviates the need for RNA purification reagents. It could enable wider use of Covid-19 testing for diagnosis, screening, and research in countries and regions where laboratory capabilities are limiting.

Rapid Direct Nucleic Acid Amplification Test without RNA Extraction for SARS-CoV-2 Using a Portable PCR Thermocycler

Rapid Direct Nucleic Acid Amplification Test without RNA Extraction for SARS-CoV-2 Using a Portable PCR Thermocycler.

Coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The rapid, sensitive and specific diagnosis of SARS-CoV-2 by fast and unambiguous testing is widely recognized to be critical in responding to the ongoing outbreak. Since the current testing capacity of RT-PCR-based methods is being challenged due to the extraordinary demand of supplies, such as RNA extraction kits and PCR reagents worldwide, alternative and/or complementary testing assays should be developed. Here, we exploit the potential of mass spectrometry technology combined with machine learning algorithms as an alternative fast tool for SARS-CoV-2 detection from nasopharyngeal swabs samples. According to our preliminary results, mass spectrometry-based methods combined with multivariate analysis showed an interesting potential as a complementary diagnostic tool and further steps should be focused on sample preparation protocols and the improvement of the technology applied.

A Combined approach of MALDI-TOF Mass Spectrometry and multivariate analysis as a potential tool for the detection of SARS-CoV-2 virus in nasopharyngeal swabs

Summary The COVID-19 pandemic is a global public health crisis. Significant delays in the rapid development and distribution of diagnostic testing for SARS-CoV-2 infection have prevented adequate public health management of the disease, impacting the timely mapping of viral spread and the conservation of personal protective equipment. Furthermore, vulnerable populations such as those served by Boston Medical Center (BMC), the largest safety net hospital in New England, represent a high-risk group across multiple dimensions, including a higher prevalence of pre-existing conditions and substance use disorders, lower health maintenance, unstable housing, and a propensity for rapid community spread, highlighting the urgent need for expedient and reliable in-house testing. Here, we report the implementation of a SARS-CoV-2 diagnostic RT-PCR assay with rapid turnaround time enabling more informed decisions with personal and public health ramifications. This work provides a blueprint for academic centers and community hospitals lacking capital-intensive automated laboratory machinery to implement in-house testing.

Rapid Implementation of a SARS-CoV-2 Diagnostic qRT-PCR Test with Emergency Use Authorization at a Large Academic Safety-Net Hospital

Diagnosis of persons exposed to/infected with severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) is central to controlling the global pandemic of COVID-19. Currently, several diagnostic modalities are available for COVID-19, each with its own pros and cons. Although there is a global consensus to increase the testing capacity, it is also essential to prudently utilize these tests to control the pandemic. In this paper, we have reviewed the current array of diagnostics for SARS-CoV-2, highlighted the gaps in current diagnostic modalities, and their role in community surveillance and control of the pandemic. The different modalities of COVID-19 diagnosis discussed are: clinical and radiological, molecular based (laboratory based and point-of-care), Immunoassay based (ELISA, rapid antigen and antibody detection tests) and digital diagnostics (artificial intelligence based algorithms). The role of rapid antigen/antibody detection tests in community surveillance has also been described here. These tests can be used to identify asymptomatic persons exposed to the virus and in community based seroprevalence surveys to assess the epidemiology of spread of the virus. However, there are few concerns about the accuracy of these tests which needs to evaluated beforehand.

COVID 19 diagnostic multiplicity and its role in community surveillance and control.

The ongoing epidemic of caused by the coronavirus SARS-CoV-2 starting in December 2019 poses a serious public health threat globally. The virus is highly infectious and transmitted mainly through droplets and contacts, and is associated with a high risk of pneumonia. A small number of patients may present with acute respiratory distress syndrome with severe respiratory complications, which can lead even to death. The selection of appropriate detection techniques and methods for accurate and rapid identification of pathogens therefore plays a key role in improving the diagnosis and treatment of the patients and containing the outbreak. In this review, the authors gives an overview of the virus laboratory detection technology, including virus isolation and culture, real-time fluorescent PCR, gene sequencing, serological antibody detection, and the gene editing technology based on CRISPR/Cas13 system. These techniques are expected to provide valuable assistance in controlling the epidemic and new ideas for future researches.

[Laboratory testing techniques for SARS-CoV-2]

When South Florida became a hotspot for COVID-19 disease in March 2020, we faced an urgent need to develop test capability to detect SARS-CoV-2 infection. We assembled a transdisciplinary team of knowledgeable and dedicated physicians, scientists, technologists and administrators, who rapidly built a multi-platform, PCR- and serology- based detection program, established drive-thru facilities and drafted and implemented guidelines that enabled efficient testing of our patients and employees. This process was extremely complex, due to the limited availability of needed reagents, but outreach to our research scientists and to multiple diagnostic laboratory companies and government officials enabled us to implement both FDA authorized and laboratory developed testing (LDT)-based testing protocols. We analyzed our workforce needs and created teams of appropriately skilled and certified workers, to safely process patient samples and conduct SARS-CoV-2 testing and contact tracing. We initiated smart test ordering, interfaced all testing platforms with our electronic medical record, and went from zero testing capacity, to testing hundreds of healthcare workers and patients daily, within three weeks. We believe our experience can inform the efforts of others, when faced with a crisis situation.

A how-to-guide to building a robust SARS-CoV-2 testing program at a university-based health system

Timely detection and diagnosis are urgently needed to guide epidemiological measures, infection control, antiviral treatment, and vaccine research. In this review, biomarkers/indicators for diagnosis of coronavirus disease 2019 (COVID-19) or detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the environment are summarized and discussed. It is concluded that the detection methods targeting antibodies are not suitable for screening of early and asymptomatic cases since most patients had an antibody response at about 10 days after onset of symptoms. However, antibody detection methods can be combined with quantitative real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) to significantly improve the sensitivity and specificity of diagnosis, and boost vaccine research. Fast, sensitive and accurate detection methods targeting antigens need to be developed urgently. Various specimens for diagnosis or detection are compared and analyzed. Among them, deep throat saliva and induced sputum are desired for RT-qPCR test or other early detection technologies. Chest computerized tomography (CT) scan, RT-qPCR, lateral flow immunochromatographic strip (LFICS) for diagnosis of COVID-19 are summarized and compared. Specially, potential electrochemical biosensor, surface enhanced Raman scattering (SERS)-based biosensor, and artificial intelligence (AI) assisted diagnosis of COVID-19 are emphasized. Finally, some commercialized portable detection device, current challenges and future directions are discussed.

Diagnostic methods and potential portable biosensors for coronavirus disease 2019

Background The SARS-CoV-2 virus is responsible for the infectious respiratory disease called COVID-19 (COronaVIrus Disease). In response to the growing COVID-19 pandemic, Rapid Diagnostic Tests (RDTs) have been developed to detect specific antibodies, IgG and IgM, to SARS-CoV-2 virus in human whole blood. We conducted a real-life study to evaluate the performance of two RDTs, COVID-PRESTO and COVID-DUO, compared to the gold standard, RT-PCR. Methods RT-PCR testing of SARS-Cov-2 was performed from nasopharyngeal swab specimens collected in adult patients visiting the infectious disease department at the hospital (Orleans, France). Fingertip whole blood samples taken at different time points after onset of the disease were tested with RDTs. The specificity and sensitivity of the rapid test kits compared to test of reference (RT-PCR) were calculated. Results Among 381 patients with symptoms of COVID-19 who went to the hospital for a diagnostic, 143 patients were RT-PCR negative. Results of test with RDTs were all negative for these patients, indicating a specificity of 100% for both RDTs. In the RT-PCR positive subgroup (n=238), 133 patients were tested with COVID-PRESTO and 129 patients were tested with COVID-DUO (24 patients tested with both). The further the onset of symptoms was from the date of collection, the greater the sensitivity. The sensitivity of COVID-PRESTO test ranged from 10.00% for patients having experienced their 1st symptoms from 0 to 5 days ago to 100% in patients where symptoms had occurred more than 15 days before the date of tests. For COVID-DUO test, the sensitivity ranged from 35.71% [0-5 days] to 100% (> 15 days). Conclusion COVID-PRESTO and DUO RDTs turned out to be very specific (none false positive) and to be sensitive enough after 15 days from onset of symptom. These easy to use IgG/IgM combined test kits are the first ones allowing a screening with capillary blood sample, by typing from a finger prick. These rapid tests are particularly interesting for screening in low resource settings.

Evaluation of performance of two SARS-CoV-2 Rapid whole-blood finger-stick IgM-IgGCombined Antibody Tests

Given the scale and rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, known as 2019-nCov) infection (COVID-19), the ongoing global SARS-CoV-2 outbreak has become a huge public health issue. Rapid and precise diagnostic methods are thus immediately needed for diagnosing COVID-19, providing timely treatment and facilitating infection control. A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) coupled with nanoparticles-based biosensor (NBS) assay (RT-LAMP-NBS) was successfully established for rapidly and accurately diagnosing COVID-19. A simple equipment (such as heating block) was required for maintaining a constant temperature (63 C) for only 40 min. Using two designed LAMP primer sets, F1ab (opening reading frame 1a/b) and np (nucleoprotein) genes of SARS-CoV-2 were simultaneously amplified and detected in a one-step and single-tube reaction, and the detection results were easily interpreted by NBS. The sensitivity of SARS-CoV-2 RT-LAMP-NBS was 12 copies (each of detection target) per reaction, and no cross-reactivity was generated from non-SARS-CoV-2 templates. Among clinically diagnosed COVID-19 patients, the analytical sensitivity of SARS-CoV-2 was 100% (33/33) in the oropharynx swab samples, and the assay's specificity was also 100% (96/96) when analyzed the clinical samples collected from non-COVID-19 patients. The total diagnosis test from sample collection to result interpretation only takes approximately 1 h. In sum, the RT-LAMP-NBS is a promising tool for diagnosing the current SARS-CoV-2 infection in first line field, public health and clinical laboratories, especially for resource-challenged regions.

Reverse transcription loop-mediated isothermal amplification combined with nanoparticles-based biosensor for diagnosis of COVID-19

Diagnostic tests for the coronavirus infection 2019 (COVID-19) are critical for prompt diagnosis, treatment and isolation to break the cycle of transmission A positive real-time reverse-transcriptase polymerase chain reaction (RT-PCR), in conjunction with clinical and epidemiologic data, is the current standard for diagnosis, but several challenges still exist Serological assays help to understand epidemiology better and to evaluate vaccine responses but they are unreliable for diagnosis in the acute phase of illness or assuming protective immunity Serology is gaining attention, mainly because of convalescent plasma gaining importance as treatment for clinically worsening COVID-19 patients We provide a narrative review of peer-reviewed research studies on RT-PCR, serology and antigen immune-assays for COVID-19, briefly describe their lab methods and discuss their limitations for clinical practice

Laboratory Tests for COVID-19: A Review of Peer-Reviewed Publications and Implications for Clinical UIse

The COVID-19 pandemic is a global health emergency characterized by the high rate of transmission and ongoing increase of cases globally. Rapid point-of-care (PoC) diagnostics to detect the causative virus, SARS-CoV-2, are urgently needed to identify and isolate patients, contain its spread and guide clinical management. In this work, we report the development of a rapid PoC diagnostic test (< 20 min) based on reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) and semiconductor technology for the detection of SARS-CoV-2 from extracted RNA samples. The developed LAMP assay was tested on a real-time benchtop instrument (RT-qLAMP) showing a lower limit of detection of 10 RNA copies per reaction. It was validated against 183 clinical samples including 127 positive samples (screened by the CDC RT-qPCR assay). Results showed 90.55% sensitivity and 100% specificity when compared to RT-qPCR and average positive detection times of 15.45 {+/-} 4.43 min. For validating the incorporation of the RT-LAMP assay onto our PoC platform (RT-eLAMP), a subset of samples was tested (n=40), showing average detection times of 12.89 {+/-} 2.59 min for positive samples (n=34), demonstrating a comparable performance to a benchtop commercial instrument. Paired with a smartphone for results visualization and geo-localization, this portable diagnostic platform with secure cloud connectivity will enable real-time case identification and epidemiological surveillance.

A handheld point-of-care system for rapid detection of SARS-CoV-2 in under 20 minutes

A new and rapid approach for detecting COVID-19 based on S1 protein fragments

Abstract Laboratory-based diagnostic measures including virological and serological tests are essential for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Real-time reverse transcription-polymerase chain reactions (rRT-PCR) can detect SARS-COV-2 by targeting open reading frame-1 antibodies (ORF1ab), envelope protein, nucleocapsid protein, RNA-dependent RNA polymerase genes, and the N1, N2, and N3 (3N) target genes. Therefore, rRT-PCR remains the primary method of diagnosing SARS-CoV-2 despite being limited by false-negative results, long turnaround, complex protocols, and a need for skilled personnel. Serological diagnosis of coronavirus disease 2019 (COVID-19) is simple and does not require complex techniques and equipment, rendering it suitable for rapid detection and massive screening. However, serological tests cannot confirm SARS-CoV-2, and results will be false-negative when antibody concentrations fall below detection limits. Balancing the increased use of laboratory tests, risk of testing errors, need for tests, burden on healthcare systems, benefits of early diagnosis, and risk of unnecessary exposure is a significant and persistent challenge in diagnosing COVID-19.

In vitro diagnostics of coronavirus disease 2019: technologies and application

[Laboratory testing techniques for SARS-CoV-2].

The outbreak of COVID-19 has taken a large number of lives since 2019 and the death toll continues to increase all over the world. Recent data reports that about 27 lacs of people are infected with this virus till date and around 2 lacs are dead due to this pandemic. The situation in India is no way better. In India, almost all the states have become victim of this deadly pandemic. Considering the enormous population in India, citizens here are facing acute shortage of detection kits and many are dying even before the knowledge of their infection. The present treatise proposes a molecularly imprinted polymer (MIP) based technique for simple and rapid detection of COVID-19. The technique will be inexpensive, selective, reusable and easy to handle. It has been already implemented in our laboratory in order to detect the taste contributing agents found in tea. This article discusses the detailed methodology and the resultant analytical characteristic of the sensors developed so far and also outlines the suitability of the MIP technique towards fabrication of testing kits for rapid detection of COVID-19.

A Molecularly Imprinted Polymer-Based Technology for Rapid Testing of COVID-19

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the need for different types of diagnostics, comparative validation of new tests, faster approval by federal agencies, and rapid production of test kits to meet global demands. In this Perspective, we discuss the utility and challenges of current diagnostics for COVID-19.

COVID-19 diagnostics in context

Abstract Recently, a novel coronavirus (SARS-CoV-2; coronavirus disease 2019, COVID-19) has emerged, rapidly spreading and severely straining the capacity of the global health community. Many nations are employing combinations of containment and mitigation strategies, where early diagnosis of COVID-19 is vital in controlling illness progression and limiting viral spread within the population. Thus, rapid and accurate methods of early detection are vital to contain COVID-19 and prevent further spread and predicted subsequent infectious waves of viral recurrence in future. Immediately after its initial characterization, Chinese and American Centers for Disease Control and Prevention (CDCs) rapidly employed molecular assays for detection of COVID-19, mostly employing real-time polymerase chain reaction (RT-PCR) methods. However, such methods require specific expensive items of equipment and highly trained analysts, requiring upwards of 4-8 hours to process. These requirements coupled with associated financial pressures may prevent effective deployment of such diagnostic tests. Loop mediated isothermal amplification(LAMP) is method of nucleic acid amplification which exhibits increased sensitivity and specificity are significantly rapid, and do not require expensive reagents or instruments, which aids in cost reduction for coronavirus detection. Studies have shown the successful application of LAMP assays in various forms to detect coronavirus RNA in patient samples, demonstrating that 1-10 copies of viral RNA template per reaction are sufficient for successful detection, ∼100-fold more sensitive than conventional RT-PCR methods. Importantly, studies have also now demonstrated the effectiveness of LAMP methodology in the detection of SARS-CoV-2 RNA at significantly low levels, particularly following numerous improvements to LAMP assay protocols. We hypothesise that recent advancements in enhanced LAMP protocols assay perhaps represent the best chance for a rapid and robust assay for field diagnosis of COVID-19, without the requirement of specialized equipment and highly trained professionals to interpret results. Herein, we present our arguments with a view to disseminate such findings, to assist the combat of this virus that is proving so devastating. We hope that this strategy could be applied rapidly, and confirmed for viability with clinical samples, before being rolled out for mass-diagnostic testing in these current times.

Loop mediated isothermal amplification (LAMP) assays as a rapid diagnostic for COVID-19

COVID-19 has resulted in a global health crisis that may become even more acute over the upcoming months. One of the main reasons behind the current rapid growth of COVID-19 in the U.S. population is the limited availability of testing kits and the relatively-high cost of screening tests. In this draft, we demonstrate the effectiveness of group testing (pooling) ideas to accelerate testing for COVID-19. This draft is semi-tutorial in nature and is written for a broad audience with interest in mathematical formulations relevant to COVID-19 testing. Therefore, ideas are presented through illustrative examples rather than through purely theoretical formulations. The focus is also on pools of size less than 64 such as what is practical with current RT-PCR technology.

On Accelerated Testing for COVID-19 Using Group Testing

OBJECTIVES: In the context of the Covid-19 pandemic, the development and validation of rapid and easy-to-perform diagnostic methods are of high priority. We evaluated a novel rapid antigen detection test (RDT) for SARS-CoV-2 in respiratory samples. METHODS: The fluorescence immunochromatographic SARS-CoV-2 antigen test (Bioeasy Biotechnology Co., Shenzhen, China) was evaluated using universal transport medium with nasopharyngeal (NP) and oropharyngeal (OP) swabs from suspected Covid-19 cases. Diagnostic accuracy was determined in comparison to SARS-CoV-2 real time (RT)-PCR. RESULTS: A total of 127 samples were included; 82 were RT-PCR positive. Median patients' age was 38 years, 53.5% were male, and 93.7% were from the first week after symptom onset. Overall sensitivity and specificity were 93.9% (CI95% 86.5-97.4) and 100% (CI95% 92.1-100), respectively, with a diagnostic accuracy of 96.1% and Kappa coefficient of 0.9. Sensitivity was significantly higher in samples with high viral loads. CONCLUSIONS: The evaluated RDT showed a high sensitivity and specificity in samples mainly obtained during the first week of symptoms and with high viral loads, despite the use of a non-validated sample material. The assay has the potential to become an important tool for early diagnosis of SARS-CoV-2, particularly in situations with limited access to molecular methods.

Evaluation of novel antigen-based rapid detection test for the diagnosis of SARS-CoV-2 in respiratory samples

There is an ongoing worldwide coronavirus disease 2019 (Covid-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At present, confirmatory diagnosis is by reverse transcription polymerase chain reaction (RT-PCR), typically taking several hours and requiring a molecular laboratory to perform. There is an urgent need for rapid, simplified and cost-effective detection methods. We have developed and analytically validated a protocol for direct rapid extraction-free PCR (DIRECT-PCR) detection of SARS-CoV-2 without the need for nucleic acid purification. As few as 6 RNA copies per reaction of viral nucleocapsid (N) gene from respiratory samples such as sputum and nasal exudate can be detected directly using our one-step inhibitor-resistant assay. The performance of this assay was validated on a commercially available portable PCR thermocycler. Viral lysis, reverse transcription, amplification and detection are achieved in a single-tube homogeneous reaction within 36 minutes. This minimized hands-on time, reduces turnaround-time for sample-to-result and obviates the need for RNA purification reagents. It could enable wider use of Covid-19 testing for diagnosis, screening and research in countries and regions where laboratory capabilities are limiting.

Rapid direct nucleic acid amplification test without RNA extraction for SARS-CoV-2 using a portable PCR thermocycler

COVID-19 is the most rapidly growing pandemic in modern time, and the need for serological testing is most urgent. Although the diagnostics of acute patients by RT-PCR is both efficient and specific, we are also crucially in need of serological tools for investigating antibody responses and assessing individual and potential herd immunity. We evaluated a commercially available test developed for rapid (within 15 minutes) detection of SARS-CoV-2-specific IgM and IgG by 29 PCR-confirmed COVID-19 cases and 124 negative controls. The results revealed a sensitivity of 69% and 93.1% for IgM and IgG, respectively, based solely on PCR-positivity due to the absence of a serological gold standard. The assay specificities were shown to be 100% for IgM and 99.2% for IgG. This indicates that the test is suitable for assessing previous virus exposure, although negative results may be unreliable during the first weeks after infection. More detailed studies on antibody responses during and post infection are urgently needed.

Evaluation of a COVID-19 IgM and IgG rapid test; an efficient tool for assessment of past exposure to SARS-CoV-2

OBJECTIVES: In the context of the Covid-19 pandemic, the development and validation of rapid and easy-to-perform diagnostic methods are of high priority. We evaluated a novel rapid antigen detection test (RDT) for SARS-CoV-2 in respiratory samples. METHODS: The fluorescence immunochromatographic SARS-CoV-2 antigen test (Bioeasy Biotechnology Co., Shenzhen, China) was evaluated using universal transport medium with nasopharyngeal (NP) and oropharyngeal (OP) swabs from suspected Covid-19 cases. Diagnostic accuracy was determined in comparison to SARS-CoV-2 real time (RT)-PCR. RESULTS: A total of 127 samples were included; 82 were RT-PCR positive. Median patients’ age was 38 years, 53.5% were male, and 93.7% were from the first week after symptom onset. Overall sensitivity and specificity were 93.9% (CI95% 86.5–97.4) and 100% (CI95% 92.1–100), respectively, with a diagnostic accuracy of 96.1% and Kappa coefficient of 0.9. Sensitivity was significantly higher in samples with high viral loads. CONCLUSIONS: The evaluated RDT showed a high sensitivity and specificity in samples mainly obtained during the first week of symptoms and with high viral loads, despite the use of a non-validated sample material. The assay has the potential to become an important tool for early diagnosis of SARS-CoV-2, particularly in situations with limited access to molecular methods.

Recently, a novel coronavirus (SARS-CoV-2; coronavirus disease 2019, COVID-19) has emerged, rapidly spreading and severely straining the capacity of the global health community. Many nations are employing combinations of containment and mitigation strategies, where early diagnosis of COVID-19 is vital in controlling illness progression and limiting viral spread within the population. Thus, rapid and accurate methods of early detection are vital to contain COVID-19 and prevent further spread and predicted subsequent infectious waves of viral recurrence in future. Immediately after its initial characterization, Chinese and American Centers for Disease Control and Prevention (CDCs) rapidly employed molecular assays for detection of COVID-19, mostly employing real-time polymerase chain reaction (RT-PCR) methods. However, such methods require specific expensive items of equipment and highly trained analysts, requiring upwards of 4-8 h to process. These requirements coupled with associated financial pressures may prevent effective deployment of such diagnostic tests. Loop mediated isothermal amplification(LAMP) is method of nucleic acid amplification which exhibits increased sensitivity and specificity are significantly rapid, and do not require expensive reagents or instruments, which aids in cost reduction for coronavirus detection. Studies have shown the successful application of LAMP assays in various forms to detect coronavirus RNA in patient samples, demonstrating that 1-10 copies of viral RNA template per reaction are sufficient for successful detection, ~100-fold more sensitive than conventional RT-PCR methods. Importantly, studies have also now demonstrated the effectiveness of LAMP methodology in the detection of SARS-CoV-2 RNA at significantly low levels, particularly following numerous improvements to LAMP assay protocols. We hypothesise that recent advancements in enhanced LAMP protocols assay perhaps represent the best chance for a rapid and robust assay for field diagnosis of COVID-19, without the requirement of specialized equipment and highly trained professionals to interpret results. Herein, we present our arguments with a view to disseminate such findings, to assist the combat of this virus that is proving so devastating. We hope that this strategy could be applied rapidly, and confirmed for viability with clinical samples, before being rolled out for mass-diagnostic testing in these current times.

Abstract The ongoing pandemic of SARS-CoV-2 is a one of the most devastating outbreaks witnessed in the last 100 years. The outbreak started in China's hinterland and spread rapidly to almost every country culminating in woefully overwhelmed healthcare systems in most countries. The only approved diagnostic test to accompany radiographic evaluation is the reverse-transcriptase PCR. However, the applicability of this test in diagnosis and surveillance is challenged by global shortage in reagents and unavailability of well-equipped laboratories with specialized staff in several low- and middle-income countries. The need for development of accurate and rapid diagnostic assays became apparent. Handful of immunodiagnostic tests and other molecular approaches were developed and tested. Other recently developed point-of-care molecular tests are expected to be helpful in pandemic management since no particular skills are required from the operator. Fortunately, handful of serological tests have granted authorization to be used under emergency situation by FDA in diagnosis of SARS-CoV-2.

Laboratory diagnosis of SARS-CoV-2: available approaches and limitations

Rapid detection of COVID-19 coronavirus using a reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) diagnostic platform