Improving Performance of a SARS-CoV-2 RT-LAMP Assay for Use With a Portable Isothermal Fluorimeter: Towards a Point-of-Care Molecular Testing Strategy

AbstractAlthough the most effective strategy for preventing or containing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreaks relies on early diagnosis, the paramount and unprecedented number of tests needed to fully achieve this target is overwhelming worldwide testing supply and capacity. Molecular detection of SARS-CoV-2 RNA in nasopharyngeal swabs is still considered the reference diagnostic approach. Nonetheless, identification of SARS-CoV-2 proteins in upper respiratory tract specimens and/or saliva by means of rapid (antigen) immunoassays is emerging as a promising screening approach. These tests have some advantages compared to molecular analysis, such as point of care availability, no need of skilled personnel and dedicated instrumentation, lower costs and short turnaround time. However, these advantages are counterbalanced by lower diagnostic sensitivity compared to molecular testing, which would only enable to identifying patients with higher SARS-CoV-2 viral load. The evidence accumulated to-date has hence persuaded us to develop a tentative algorithm, which would magnify the potential benefits of rapid antigen testing in SARS-CoV-2 diagnostics.

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Screening, Diagnostic and Prognostic Tests for COVID-19: A Comprehensive Review

Life ◽

10.3390/life11060561 ◽

2021 ◽

Vol 11 (6) ◽

pp. 561

Author(s):

Mariana Ulinici ◽

Serghei Covantev ◽

James Wingfield-Digby ◽

Apostolos Beloukas ◽

Alexander G. Mathioudakis ◽

...

Keyword(s):

Point Of Care ◽

Standard Test ◽

Current Evidence ◽

Molecular Testing ◽

Rt Pcr ◽

Diagnostic Screening ◽

Comprehensive Review ◽

Prognostic Tests ◽

Polymerase Chain ◽

Antigen Testing

While molecular testing with real-time polymerase chain reaction (RT-PCR) remains the gold-standard test for COVID-19 diagnosis and screening, more rapid or affordable molecular and antigen testing options have been developed. More affordable, point-of-care antigen testing, despite being less sensitive compared to molecular assays, might be preferable for wider screening initiatives. Simple laboratory, imaging and clinical parameters could facilitate prognostication and triage. This comprehensive review summarises current evidence on the diagnostic, screening and prognostic tests for COVID-19.

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N-protein presents early in blood, dried blood and saliva during asymptomatic and symptomatic SARS-CoV-2 infection

Nature Communications ◽

10.1038/s41467-021-22072-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Dandan Shan ◽

Joseph M. Johnson ◽

Syrena C. Fernandes ◽

Hannah Suib ◽

Soyoon Hwang ◽

...

Keyword(s):

Single Molecule ◽

High Performance ◽

Point Of Care ◽

Capillary Blood ◽

Molecular Testing ◽

Sample Collection ◽

Protein Assay ◽

N Protein ◽

Protein Levels ◽

Percent Agreement

AbstractThe COVID-19 pandemic continues to have an unprecedented impact on societies and economies worldwide. There remains an ongoing need for high-performance SARS-CoV-2 tests which may be broadly deployed for infection monitoring. Here we report a highly sensitive single molecule array (Simoa) immunoassay in development for detection of SARS-CoV-2 nucleocapsid protein (N-protein) in venous and capillary blood and saliva. In all matrices in the studies conducted to date we observe >98% negative percent agreement and >90% positive percent agreement with molecular testing for days 1–7 in symptomatic, asymptomatic, and pre-symptomatic PCR+ individuals. N-protein load decreases as anti-SARS-CoV-2 spike-IgG increases, and N-protein levels correlate with RT-PCR Ct-values in saliva, and between matched saliva and capillary blood samples. This Simoa SARS-CoV-2 N-protein assay effectively detects SARS-CoV-2 infection via measurement of antigen levels in blood or saliva, using non-invasive, swab-independent collection methods, offering potential for at home and point of care sample collection.

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LAMP-based foldable microdevice platform for the rapid detection of Magnaporthe oryzae and Sarocladium oryzae in rice seed

Scientific Reports ◽

10.1038/s41598-020-80644-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

M. K. Prasannakumar ◽

P. Buela Parivallal ◽

Devanna Pramesh ◽

H. B. Mahesh ◽

Edwin Raj

Keyword(s):

Magnaporthe Oryzae ◽

Plant Pathogens ◽

Point Of Care ◽

Lamp Assay ◽

Rice Seed ◽

Highly Sensitive ◽

Polymerase Chain ◽

Rice Seeds ◽

Template Dna ◽

Assay Conditions

AbstractRice blast (caused by Magnaporthe oryzae) and sheath rot diseases (caused by Sarocladium oryzae) are the most predominant seed-borne pathogens of rice. The detection of both pathogens in rice seed is essential to avoid production losses. In the present study, a microdevice platform was designed, which works on the principles of loop-mediated isothermal amplification (LAMP) to detect M. oryzae and S. oryzae in rice seeds. Initially, a LAMP, polymerase chain reaction (PCR), quantitative PCR (qPCR), and helicase dependent amplification (HDA) assays were developed with primers, specifically targeting M. oryzae and S. oryzae genome. The LAMP assay was highly efficient and could detect the presence of M. oryzae and S. oryzae genome at a concentration down to 100 fg within 20 min at 60 °C. Further, the sensitivity of the LAMP, HDA, PCR, and qPCR assays were compared wherein; the LAMP assay was highly sensitive up to 100 fg of template DNA. Using the optimized LAMP assay conditions, a portable foldable microdevice platform was developed to detect M. oryzae and S. oryzae in rice seeds. The foldable microdevice assay was similar to that of conventional LAMP assay with respect to its sensitivity (up to 100 fg), rapidity (30 min), and specificity. This platform could serve as a prototype for developing on-field diagnostic kits to be used at the point of care centers for the rapid diagnosis of M. oryzae and S. oryzae in rice seeds. This is the first study to report a LAMP-based foldable microdevice platform to detect any plant pathogens.

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INSIGHT: A population-scale COVID-19 testing strategy combining point-of-care diagnosis with centralized high-throughput sequencing

Science Advances ◽

10.1126/sciadv.abe5054 ◽

2021 ◽

Vol 7 (7) ◽

pp. eabe5054

Author(s):

Qianxin Wu ◽

Chenqu Suo ◽

Tom Brown ◽

Tengyao Wang ◽

Sarah A. Teichmann ◽

...

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Limit Of Detection ◽

Point Of Care ◽

Population Level ◽

Reaction Products ◽

Testing Strategy ◽

Asymptomatic Patients ◽

Testing Stage ◽

Population Scale

We present INSIGHT [isothermal NASBA (nucleic acid sequence–based amplification) sequencing–based high-throughput test], a two-stage coronavirus disease 2019 testing strategy, using a barcoded isothermal NASBA reaction. It combines point-of-care diagnosis with next-generation sequencing, aiming to achieve population-scale testing. Stage 1 allows a quick decentralized readout for early isolation of presymptomatic or asymptomatic patients. It gives results within 1 to 2 hours, using either fluorescence detection or a lateral flow readout, while simultaneously incorporating sample-specific barcodes. The same reaction products from potentially hundreds of thousands of samples can then be pooled and used in a highly multiplexed sequencing–based assay in stage 2. This second stage confirms the near-patient testing results and facilitates centralized data collection. The 95% limit of detection is <50 copies of viral RNA per reaction. INSIGHT is suitable for further development into a rapid home-based, point-of-care assay and is potentially scalable to the population level.

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Clinical assessment of the Roche SARS-CoV-2 rapid antigen test

Diagnosis ◽

10.1515/dx-2020-0154 ◽

2021 ◽

Vol 0 (0) ◽

Author(s):

Gian Luca Salvagno ◽

Gianluca Gianfilippi ◽

Damiano Bragantini ◽

Brandon M. Henry ◽

Giuseppe Lippi

Keyword(s):

Clinical Assessment ◽

Clinical Performance ◽

Point Of Care ◽

High Viral Load ◽

Rapid Screening ◽

Clinical Samples ◽

Molecular Testing ◽

Antigen Test ◽

Community Screening ◽

Final Study

Abstract Objectives Novel point-of-care antigen assays present a promising opportunity for rapid screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. The purpose of this study was the clinical assessment of the new Roche SARS-CoV-2 Rapid Antigen Test. Methods The clinical performance of Roche SARS-CoV-2 Rapid Antigen Test was evaluated vs. a reverse transcription polymerase chain reaction (RT-PCR) laboratory-based assay (Seegene AllplexTM2019-nCoV) in nasopharyngeal swabs collected from a series of consecutive patients referred for SARS-CoV-2 diagnostics to the Pederzoli Hospital (Peschiera del Garda, Verona, Italy) over a 2-week period. Results The final study population consisted of 321 consecutive patients (mean age, 46 years and IQR, 32–56 years; 181 women, 56.4%), with 149/321 (46.4%) positive for SARS-CoV-2 RNA via the Seegene AllplexTM2019-nCoV Assay, and 109/321 (34.0%) positive with Roche SARS-CoV-2 Rapid Antigen Test, respectively. The overall accuracy of Roche SARS-CoV-2 Rapid Antigen Test compared to molecular testing was 86.9%, with 72.5% sensitivity and 99.4% specificity. Progressive decline in performance was observed as cycle threshold (Ct) values of different SARS-CoV-2 gene targets increased. The sensitivity was found to range between 97–100% in clinical samples with Ct values <25, between 50–81% in those with Ct values between 25 and <30, but low as 12–18% in samples with Ct values between 30 and <37. Conclusions The clinical performance of Roche SARS-CoV-2 Rapid Antigen Test is excellent in nasopharyngeal swabs with Ct values <25, which makes it a reliable screening test in patients with high viral load. However, mass community screening would require the use of more sensitive techniques.

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Recent advances and novel approaches in laboratory-based diagnostic mycology

Medical Mycology ◽

10.1093/mmy/myy159 ◽

2019 ◽

Vol 57 (Supplement_3) ◽

pp. S259-S266 ◽

Cited By ~ 4

Author(s):

P Lewis White

Keyword(s):

Point Of Care ◽

Antibody Detection ◽

Molecular Testing ◽

Point Of Care Testing ◽

Proximity Ligation ◽

Molecular Assays ◽

Lateral Flow Assays ◽

Novel Approaches ◽

Exciting Time ◽

Generation Sequencing

Abstract The field of diagnostic mycology represents much more than culture and microscopy and is rapidly embracing novel techniques and strategies to help overcome the limitations of conventional approaches. Commercial molecular assays increase the applicability of PCR testing and may identify markers of antifungal resistance, which are of great clinical concern. Lateral flow assays simplify testing and turn-around time, with potential for point of care testing, while proximity ligation assays embrace the sensitivity of molecular testing with the specificity of antibody detection. The first evidence of patient risk stratification is being described and together with the era of next generation sequencing represents an exciting time in mycology.

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Clinical and Economic Impact of ‘ROS1-Testing’ Strategy Compared to a ‘No- ROS1-Testing’ Strategy in Advanced NSCLC in Spain

10.21203/rs.3.rs-1103970/v1 ◽

2021 ◽

Author(s):

Federico Rojo ◽

Esther Conde ◽

Héctor Torres ◽

Luis Cabezón-Gutiérrez ◽

Dolores Bautista ◽

...

Keyword(s):

Markov Model ◽

Economic Impact ◽

Expert Opinion ◽

Advanced Nsclc ◽

Direct Costs ◽

Base Case ◽

Molecular Testing ◽

Testing Strategy ◽

Lifetime Horizon ◽

Life Years

Abstract Background: Detection of the ROS1 rearrangement is mandatory in patients with advanced or metastatic non-small cell lung cancer (NSCLC) to allow targeted therapy with specific inhibitors. However, in Spanish clinical practice ROS1 determination is not yet fully widespread. The aim of this study is to determine the clinical and economic impact of sequentially testing ROS1 in addition to EGFR and ALK in Spain.Methods: A joint model (decision-tree and Markov model) was developed to determine the cost-effectiveness of testing ROS1 strategy versus a no-ROS1 testing strategy in Spain. Distribution of ROS1 techniques, rates of testing, positivity, and invalidity of biomarkers included in the analysis (EGFR, ALK, ROS1 and PD-L1) were based on expert opinion and Lungpath real-world database. Treatment allocation depending on the molecular testing results was defined by expert opinion. For each treatment, a 3-states Markov model was developed, where progression free survival (PFS) and overall survival (OS) curves were parameterized using exponential extrapolations to model transition of patients among health states. Only medical direct costs were included (€ 2021). A lifetime horizon was considered and a discount rate of 3% was applied for both costs and effects. Both deterministic and probabilistic sensitivity analyses were performed to address uncertainty.Results: A target population of 8,755 patients with advanced NSCLC (non-squamous or never smokers squamous) entered the model. Over a lifetime horizon, the ROS1 testing scenario produced additional 157.5 life years and 121.3 quality-adjusted life years (QALYs) compared with no-ROS1 testing scenario. Total direct costs were increased up to € 2,244,737 for ROS1 testing scenario. The incremental cost-utility ratio (ICUR) was 18,514 €/QALY. Robustness of the base-case results were confirmed by the sensitivity analysis.Conclusions: Our study shows that ROS1 testing in addition to EGFR and ALK is a cost-effective strategy compared to no-ROS1 testing, and it generates more than 120 QALYs in Spain over a lifetime horizon. Despite the low prevalence of ROS1 rearrangements in NSCLC patients, the clinical and economic consequences of ROS1 testing should encourage centers to test all advanced or metastatic NSCLC (non-squamous and never-smoker squamous) patients.

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Point-of-care molecular testing and antiviral treatment of influenza in residents of homeless shelters in Seattle, WA: study protocol for a stepped-wedge cluster-randomized controlled trial

Trials ◽

10.1186/s13063-020-04871-5 ◽

2020 ◽

Vol 21 (1) ◽

Author(s):

Kira L. Newman ◽

◽

Julia H. Rogers ◽

Denise McCulloch ◽

Naomi Wilcox ◽

...

Keyword(s):

Point Of Care ◽

Antiviral Treatment ◽

Homeless Shelters ◽

Molecular Testing ◽

Symptom Duration ◽

Homeless Shelter ◽

Stepped Wedge ◽

Site Testing ◽

Cluster Randomized ◽

The Impact

Abstract Introduction Influenza is an important public health problem, but data on the impact of influenza among homeless shelter residents are limited. The primary aim of this study is to evaluate whether on-site testing and antiviral treatment of influenza in residents of homeless shelters reduces influenza spread in these settings. Methods and analysis This study is a stepped-wedge cluster-randomized trial of on-site testing and antiviral treatment for influenza in nine homeless shelter sites within the Seattle metropolitan area. Participants with acute respiratory illness (ARI), defined as two or more respiratory symptoms or new or worsening cough with onset in the prior 7 days, are eligible to enroll. Approximately 3200 individuals are estimated to participate from October to May across two influenza seasons. All sites will start enrollment in the control arm at the beginning of each season, with routine surveillance for ARI. Sites will be randomized at different timepoints to enter the intervention arm, with implementation of a test-and-treat strategy for individuals with two or fewer days of symptoms. Eligible individuals will be tested on-site with a point-of-care influenza test. If the influenza test is positive and symptom onset is within 48 h, participants will be administered antiviral treatment with baloxavir or oseltamivir depending upon age and comorbidities. Participants will complete a questionnaire on demographics and symptom duration and severity. The primary endpoint is the incidence of influenza in the intervention period compared to the control period, after adjusting for time trends. Trial registration ClinicalTrials.gov NCT04141917. Registered 28 October 2019. Trial sponsor: University of Washington.

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Group A Streptococcus Testing in Pediatrics: the Move to Point-of-Care Molecular Testing

Journal of Clinical Microbiology ◽

10.1128/jcm.01494-19 ◽

2020 ◽

Vol 58 (6) ◽

Cited By ~ 1

Author(s):

Thomas Z. Thompson ◽

Allison R. McMullen

Keyword(s):

Point Of Care ◽

Streptococcal Pharyngitis ◽

Molecular Methods ◽

Multiple Point ◽

Molecular Testing ◽

Point Of Care Testing ◽

Culture Methods ◽

Content Type ◽

Molecular Group ◽

Group A

ABSTRACT Each year, there are an estimated 11 million visits to ambulatory care centers for pharyngitis in children between the ages of 3 and 18 years. While there are many causes of pediatric pharyngitis, group A streptococcal pharyngitis represents 15 to 30% of infections and is the only cause for which treatment is recommended. Unfortunately, clinical suspicion is insufficient for the accurate diagnosis of group A streptococcal pharyngitis, and laboratory testing for confirmation of Streptococcus pyogenes infection is required to prevent complications of infection. Traditionally, throat swabs are inoculated onto agar plates for isolation of the large-zone beta-hemolytic streptococcus. However, traditional culture methods present a potential delay in treatment due to turnaround times of 18 to 48 h. In order to improve turnaround times and enhance antimicrobial stewardship, multiple point-of-care assays have been developed. This review describes current point-of-care testing for group A streptococcal pharyngitis, including rapid antigen detection tests and more recent molecular methods. Additional attention is given to the diagnostic considerations when choosing a method for group A streptococcal point-of-care testing, implementation of molecular group A streptococcal testing, and the institutional cost of immunoassays compared to those of newer molecular methods.

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