scholarly journals Colorimetric RT-LAMP SARS-CoV-2 diagnostic sensitivity relies on color interpretation and viral load

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mateus Nóbrega Aoki ◽  
Bruna de Oliveira Coelho ◽  
Luiz Gustavo Bentim Góes ◽  
Paola Minoprio ◽  
Edison Luiz Durigon ◽  
...  
Keyword(s):  
Viral Load ◽  
Reaction Time ◽  
Color Change ◽  
Rna Virus ◽  
Point Of Care ◽  
Respiratory Viruses ◽  
Lamp Reaction ◽  
Clinical Samples ◽  
Viral Loads ◽  
Naked Eye

AbstractThe use of RT-LAMP (reverse transcriptase—loop mediated isothermal amplification) has been considered as a promising point-of-care method to diagnose COVID-19. In this manuscript we show that the RT-LAMP reaction has a sensitivity of only 200 RNA virus copies, with a color change from pink to yellow occurring in 100% of the 62 clinical samples tested positive by RT-qPCR. We also demonstrated that this reaction is 100% specific for SARS-CoV-2 after testing 57 clinical samples infected with dozens of different respiratory viruses and 74 individuals without any viral infection. Although the majority of manuscripts recently published using this technique describe only the presence of two-color states (pink = negative and yellow = positive), we verified by naked-eye and absorbance measurements that there is an evident third color cluster (orange), in general related to positive samples with low viral loads, but which cannot be defined as positive or negative by the naked eye. Orange colors should be repeated or tested by RT-qPCR to avoid a false diagnostic. RT-LAMP is therefore very reliable for samples with a RT-qPCR Ct < 30 being as sensitive and specific as a RT-qPCR test. All reactions were performed in 30 min at 65 °C. The use of reaction time longer than 30 min is also not recommended since nonspecific amplifications may cause false positives.

scholarly journals Essential properties and pitfalls of colorimetric Reverse Transcription Loop-mediated Isothermal Amplification as a point-of-care test for SARS-CoV-2 diagnosis

2021 ◽  
Vol 27 (1) ◽  
Author(s):  
Bruna de Oliveira Coelho ◽  
Heloisa Bruna Soligo Sanchuki ◽  
Dalila Luciola Zanette ◽  
Jeanine Marie Nardin ◽  
Hugo Manuel Paz Morales ◽  
...  
Keyword(s):  
Viral Load ◽  
Internal Control ◽  
Reverse Transcription ◽  
Color Change ◽  
Point Of Care ◽  
Colorimetric Detection ◽  
False Negative ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Loop Mediated Isothermal Amplification

Abstract Background SARS-CoV-2 Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) colorimetric detection is a sensitive and specific point-of-care molecular biology technique used to detect the virus in only 30 min. In this manuscript we have described a few nuances of the technique still not properly described in the literature: the presence of three colors clusters; the correlation of the viral load with the color change; and the importance of using an internal control to avoid false-negative results. Methods To achieve these findings, we performed colorimetric RT-LAMP assays of 466 SARS-CoV-2 RT-qPCR validated clinical samples, with color quantification measured at 434 nm and 560 nm. Results First we determinate a sensitivity of 93.8% and specificity of 90.4%. In addition to the pink (negative) and yellow (positive) produced colors, we report for the first time the presence of an orange color cluster that may lead to wrong diagnosis. We also demonstrated using RT-qPCR and RT-LAMP that low viral loads are related to Ct values > 30, resulting in orange colors. We also demonstrated that the diagnosis of COVID-19 by colorimetric RT-LAMP is efficient until the fifth symptoms day when the viral load is still relatively high. Conclusion This study reports properties and indications for colorimetric RT-LAMP as point-of-care for SARS-CoV-2 diagnostic, reducing false results, interpretations and optimizing molecular diagnostics tests application.

scholarly journals Development of robust isothermal RNA amplification assay for lab-free testing of RNA viruses

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Radhika Biyani ◽  
Kirti Sharma ◽  
Kenji Kojima ◽  
Madhu Biyani ◽  
Vishnu Sharma ◽  
...  
Keyword(s):  
Rna Virus ◽  
Point Of Care ◽  
Human Saliva ◽  
Nucleic Acid Amplification ◽  
Potential Game ◽  
One Pot ◽  
Viral Loads ◽  
Copy Numbers ◽  
Amplification Assay ◽  
Primary Focus

AbstractSimple tests of infectiousness that return results in minutes and directly from samples even with low viral loads could be a potential game-changer in the fight against COVID-19. Here, we describe an improved isothermal nucleic acid amplification assay, termed the RICCA (RNA Isothermal Co-assisted and Coupled Amplification) reaction, that consists of a simple one-pot format of ‘sample-in and result-out’ with a primary focus on the detection of low copy numbers of RNA virus directly from saliva without the need for laboratory processing. We demonstrate our assay by detecting 16S rRNA directly from E. coli cells with a sensitivity as low as 8 CFU/μL and RNA fragments from a synthetic template of SARS-CoV-2 with a sensitivity as low as 1740 copies/μL. We further demonstrate the applicability of our assay for real-time testing at the point of care by designing a closed format for paper-based lateral flow assay and detecting heat-inactivated SARS-COV-2 virus in human saliva at concentrations ranging from 28,000 to 2.8 copies/μL with a total assay time of 15–30 min.

scholarly journals Feasibility and Effectiveness Assessment of SARS-CoV-2 Antigenic Tests in Mass Screening of a Pediatric Population and Correlation with the Kinetics of Viral Loads

Viruses ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 2071
Author(s):  
Marcello Lanari ◽  
Giovanni Battista Biserni ◽  
Matteo Pavoni ◽  
Eva Caterina Borgatti ◽  
Marta Leone ◽  
...  
Keyword(s):  
Viral Load ◽  
Mass Screening ◽  
Point Of Care ◽  
Pediatric Population ◽  
Late Phase ◽  
High Viral Load ◽  
Pediatric Emergency ◽  
Nucleic Acid Amplification ◽  
Baseline Sensitivity ◽  
Viral Loads

The gold standard for diagnosis of SARS-CoV-2 infection has been nucleic acid amplification tests (NAAT). However, rapid antigen detection kits (Ag-RDTs), may offer advantages over NAAT in mass screening, generating results in minutes, both as laboratory-based test or point-of-care (POC) use for clinicians, at a lower cost. We assessed two different POC Ag-RDTs in mass screening versus NAAT for SARS-CoV-2 in a cohort of pediatric patients admitted to the Pediatric Emergency Unit of IRCCS—Polyclinic of Sant’Orsola, Bologna (from November 2020 to April 2021). All patients were screened with nasopharyngeal swabs for the detection of SARS-CoV-2-RNA and for antigen tests. Results were obtained from 1146 patients. The COVID-19 Ag FIA kit showed a baseline sensitivity of 53.8% (CI 35.4–71.4%), baseline specificity 99.7% (CI 98.4–100%) and overall accuracy of 80% (95% CI 0.68–0.91); the AFIAS COVID-19 Ag kit, baseline sensitivity of 86.4% (CI 75.0–93.9%), baseline specificity 98.3% (CI 97.1–99.1%) and overall accuracy of 95.3% (95% CI 0.92–0.99). In both tests, some samples showed very low viral load and negative Ag-RDT. This disagreement may reflect the positive inability of Ag-RDTs of detecting antigen in late phase of infection. Among all cases with positive molecular test and negative antigen test, none showed viral loads > 106 copies/mL. Finally, we found one false Ag-RDTs negative result (low cycle thresholds; 9 × 105 copies/mL). Our results suggest that both Ag-RDTs showed good performances in detection of high viral load samples, making it a feasible and effective tool for mass screening in actively infected children.

scholarly journals Air and environmental sampling for SARS-CoV-2 around hospitalized patients with coronavirus disease 2019 (COVID-19)

2020 ◽  
Vol 41 (11) ◽  
pp. 1258-1265 ◽  
Author(s):  
Vincent Chi-Chung Cheng ◽  
Shuk-Ching Wong ◽  
Veronica Wing-Man Chan ◽  
Simon Yung-Chun So ◽  
Jonathan Hon-Kwan Chen ◽  
...  
Keyword(s):  
Viral Load ◽  
Environmental Samples ◽  
Control Measures ◽  
Clinical Samples ◽  
Contamination Rate ◽  
Median Viral Load ◽  
Air Samples ◽  
Viral Loads ◽  
Infection Control Measures ◽  
Environmental Disinfection

AbstractBackground:The role of severe respiratory coronavirus virus 2 (SARS-CoV-2)–laden aerosols in the transmission of coronavirus disease 2019 (COVID-19) remains uncertain. Discordant findings of SARS-CoV-2 RNA in air samples were noted in early reports.Methods:Sampling of air close to 6 asymptomatic and symptomatic COVID-19 patients with and without surgical masks was performed with sampling devices using sterile gelatin filters. Frequently touched environmental surfaces near 21 patients were swabbed before daily environmental disinfection. The correlation between the viral loads of patients’ clinical samples and environmental samples was analyzed.Results:All air samples were negative for SARS-CoV-2 RNA in the 6 patients singly isolated inside airborne infection isolation rooms (AIIRs) with 12 air changes per hour. Of 377 environmental samples near 21 patients, 19 (5.0%) were positive by reverse-transcription polymerase chain reaction (RT-PCR) assay, with a median viral load of 9.2 × 102 copies/mL (range, 1.1 × 102 to 9.4 × 104 copies/mL). The contamination rate was highest on patients’ mobile phones (6 of 77, 7.8%), followed by bed rails (4 of 74, 5.4%) and toilet door handles (4 of 76, 5.3%). We detected a significant correlation between viral load ranges in clinical samples and positivity rate of environmental samples (P < .001).Conclusion:SARS-CoV-2 RNA was not detectable by air samplers, which suggests that the airborne route is not the predominant mode of transmission of SARS-CoV-2. Wearing a surgical mask, appropriate hand hygiene, and thorough environmental disinfection are sufficient infection control measures for COVID-19 patients isolated singly in AIIRs. However, this conclusion may not apply during aerosol-generating procedures or in cohort wards with large numbers of COVID-19 patients.

scholarly journals A novel RT-LAMP workflow for rapid salivary diagnostics of COVID-19 and effects of age, gender and time from symptom onset

2021 ◽  
Author(s):  
Gerson Shigeru Kobayashi ◽  
Luciano Abreu Brito ◽  
Danielle De Paula Moreira ◽  
Angela May Suzuki ◽  
Gabriella Shih Ping Hsia ◽  
...  
Keyword(s):  
Viral Load ◽  
Symptom Onset ◽  
Clinical Performance ◽  
Point Of Care ◽  
Rapid Diagnostics ◽  
Rt Pcr ◽  
Point Of Care Testing ◽  
Viral Loads ◽  
Peak Viral Load ◽  
Salivary Diagnostics

Objectives: Rapid diagnostics is pivotal to curb SARS-CoV-2 transmission, and saliva has emerged as a practical alternative to naso/oropharyngeal (NOP) specimens. We aimed to develop a direct RT-LAMP workflow for viral detection in saliva, and to provide more information regarding its potential in COVID-19 diagnostics. Methods: Clinical and contrived specimens were used to screen/optimize formulations and sample processing protocols. Salivary viral load was determined in symptomatic patients to evaluate clinical performance (n = 90) and to characterize saliva based on age, gender and time from onset of symptoms (n = 49). Results: The devised workflow achieved 93.2% sensitivity, 97% specificity, and 0.895 Kappa for salivas containing >102 copies/μL. Further analyses in saliva showed peak viral load in the first days of symptoms and lower viral loads in females, particularly among young individuals (<38 years). NOP RT-PCR data did not yield relevant associations. Conclusions: This novel saliva RT-LAMP workflow can be applied to point-of-care testing. This work reinforces that saliva better correlates with transmission dynamics than NOP specimens, and reveals gender differences that may reflect higher transmission by males. To maximize detection, testing should be done immediately after symptom onset, especially in females.

scholarly journals SARS-CoV-2 infectivity correlates with high viral loads and detection of viral antigen and is terminated by seroconversion

2021 ◽  
Author(s):  
Felix Buder ◽  
Markus Bauswein ◽  
Clara L Magnus ◽  
Franz Audebert ◽  
Henriette Lang ◽  
...  
Keyword(s):  
Viral Load ◽  
Symptom Onset ◽  
Viral Antigen ◽  
Virus Isolation ◽  
Point Of Care ◽  
High Viral Load ◽  
University Hospital ◽  
Public Health Perspective ◽  
Viral Loads ◽  
Restrictive Measures

Abstract Background From a public health perspective, effective containment strategies for SARS-CoV-2 should be balanced with individual liberties. Methods We collected 79 respiratory samples from 59 patients monitored in an outpatient center or in the intensive care unit of the University Hospital Regensburg. We analyzed viral load by quantitative real-time PCR, viral antigen by point-of-care assay, time since onset of symptoms and presence of SARS-CoV-2 IgG antibodies in the context of virus isolation from respiratory specimen. Results The odds ratio for virus isolation increased 1.9-fold for each log10 level of SARS-CoV-2 RNA and 7.4-fold with detection of viral antigen, while it decreased 6.3-fold beyond 10 days of symptoms and 20.0-fold with presence of SARS-CoV-2 antibodies. The latter was confirmed for B.1.1.7 strains. The positive predictive value for virus isolation was 60.0% for viral loads above 10 7 RNA copies/mL and 50.0% for the presence of viral antigen. Symptom onset before 10 days and seroconversion predicted lack of infectivity with 93.8% and 96.0%. Conclusions Our data support quarantining patients with high viral load and detection of viral antigen, and lifting restrictive measures with increasing time to symptom onset and seroconversion. Delay of antibody formation may prolong infectivity.

scholarly journals Rapid, accurate, nucleobase detection using FnCas9

2020 ◽  
Author(s):  
Mohd. Azhar ◽  
Rhythm Phutela ◽  
Manoj Kumar ◽  
Asgar Hussain Ansari ◽  
Riya Rauthan ◽  
...  
Keyword(s):  
Point Of Care ◽  
Disease Outbreaks ◽  
Smartphone Application ◽  
Clinical Samples ◽  
Single Nucleotide Variants ◽  
Clinical Prognosis ◽  
Viral Loads ◽  
Infectious Disease Outbreaks ◽  
Dna And Rna ◽  
True Outcome

Rapid detection of pathogenic sequences or variants in DNA and RNA through a point-of-care diagnostic approach is valuable for accelerated clinical prognosis as has been witnessed during the recent COVID-19 outbreak. Traditional methods relying on qPCR or sequencing are difficult to implement in settings with limited resources necessitating the development of accurate alternative testing strategies that perform robustly. Here, we present FnCas9 Editor Linked Uniform Detection Assay (FELUDA) that employs a direct Cas9 based enzymatic readout for detecting nucleotide sequences and identifying nucleobase identity without the requirement of trans-cleavage activity of reporter molecules. We demonstrate that FELUDA is 100% accurate in detecting single nucleotide variants (SNVs) including heterozygous carriers of a mutation and present a simple design strategy in the form of a web-tool, JATAYU, for its implementation. FELUDA is semi quantitative, can be adapted to multiple signal detection platforms and can be quickly designed and deployed for versatile applications such as infectious disease outbreaks like COVID-19. Using a lateral flow readout within 1h, FELUDA shows 100% sensitivity and 97% specificity across all range of viral loads in clinical samples. In combination with RT-RPA and a smartphone application True Outcome Predicted via Strip Evaluation (TOPSE), we present a prototype for FELUDA for CoV-2 detection at home.

scholarly journals Viral load of SARS-CoV-2 across patients and compared to other respiratory viruses

2020 ◽  
Author(s):  
Damien Jacot ◽  
Gilbert Greub ◽  
Katia Jaton ◽  
Onya Opota
Keyword(s):  
Viral Load ◽  
Respiratory Viruses ◽  
Rt Pcr ◽  
Clinical Prognosis ◽  
Clinical Specimens ◽  
Viral Loads ◽  
Over Time

RT-PCR to detect SARS-CoV-2 RNA in clinical specimens was key to manage the COVID-19 pandemic. We monitored SARS-CoV-2 viral loads over time and across different patient populations. We analyzed RT-PCR results according to samples types, gender, age, and health units and compared SARS-CoV-2 viral load to other respiratory viruses, representing a total of 28,373 RT-PCR results including 22,323 SARS-CoV-2 RT-PCR. The importance of viral load to predict contagiousness and clinical prognosis is discussed.

scholarly journals Comparison of Viral Loads in Patients with Co-infections vs. Single-virus Infections

2017 ◽  
Vol 4 (suppl_1) ◽  
pp. S310-S310
Author(s):  
Stockton Beveridge ◽  
Bhinnata Piya ◽  
Laura Stewart ◽  
Mary Louise Lindegren ◽  
Tiffanie Markus ◽  
...  
Keyword(s):  
Viral Load ◽  
Respiratory Viruses ◽  
Molecular Testing ◽  
Virus Infections ◽  
Demographic Information ◽  
Research Support ◽  
Viral Pathogen ◽  
Viral Loads ◽  
Total Cohort ◽  
Co Detection

Abstract Background Molecular testing for respiratory viruses in clinical practice is common, often with multiple viruses detected. Viral load has been correlated with illness severity, but correlation of co-detection of viruses and viral load is less clear. We sought to compare cycle threshold (Ct) values, a marker inversely related to viral load, between single vs. co-detection of common respiratory viruses. Methods Children &lt;18 years with respiratory symptoms and/or fever who presented to the ED or were admitted were enrolled. Nasal/throat specimens were obtained and combined. Singleplex qRT-PCR was used to test for 11 respiratory viruses. Clinical and demographic information were collected. Results From 11/15/15-7/15/16, 1255 children were enrolled, with median age of 26.5 months, 53.4% male, 54.3% White, 38.7% Black, 6.4% other, and 23.5% Hispanic. The median days of illness were 3 days. Of the total cohort, 904 (72%) tested positive for at least one viral pathogen. Table 1compares Ct values of single vs. co-detection for each individual virus. Conclusion Single detection with RSV, HRV, AdV, and PIV had lower Ct values, indicating higher viral loads, compared with co-detection with other viruses. Additional research is needed to understand the reason for lower viral loads for co-detection vs. single detection in select respiratory viruses. Disclosures W. Schaffner, Pfizer: Scientific Advisor, Consulting fee. Merck: Scientific Advisor, Consulting fee. Novavax: Consultant, Consulting fee. Dynavax: Consultant, Consulting fee. Sanofi-pasteur: Consultant, Consulting fee. GSK: Consultant, Consulting fee. Seqirus: Consultant, Consulting fee. N. B. Halasa, sanofi pasteur: Research Contractor, Research support. Astra Zeneca: Research Contractor, Grant recipient.

scholarly journals Viral Metagenomics on Cerebrospinal Fluid

Genes ◽  
2019 ◽  
Vol 10 (5) ◽  
pp. 332 ◽  
Author(s):  
Edridge ◽  
Deijs ◽  
van Zeggeren ◽  
Kinsella ◽  
Jebbink ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Viral Load ◽  
Rna Viruses ◽  
Rna Virus ◽  
Viral Metagenomics ◽  
Clinical Samples ◽  
Cns Infections ◽  
Dnase Treatment ◽  
Low Load ◽  
Multiple Samples

Identifying the causative pathogen in central nervous system (CNS) infections is crucial for patient management and prognosis. Many viruses can cause CNS infections, yet screening for each individually is costly and time-consuming. Most metagenomic assays can theoretically detect all pathogens, but often fail to detect viruses because of their small genome and low viral load. Viral metagenomics overcomes this by enrichment of the viral genomic content in a sample. VIDISCA-NGS is one of the available workflows for viral metagenomics, which requires only a small input volume and allows multiplexing of multiple samples per run. The performance of VIDISCA-NGS was tested on 45 cerebrospinal fluid (CSF) samples from patients with suspected CNS infections in which a virus was identified and quantified by polymerase chain reaction. Eighteen were positive for an RNA virus, and 34 for a herpesvirus. VIDISCA-NGS detected all RNA viruses with a viral load >2 × 104 RNA copies/mL (n = 6) and 8 of 12 of the remaining low load samples. Only one herpesvirus was identified by VIDISCA-NGS, however, when withholding a DNase treatment, 11 of 18 samples with a herpesvirus load >104 DNA copies/mL were detected. Our results indicate that VIDISCA-NGS has the capacity to detect low load RNA viruses in CSF. Herpesvirus DNA in clinical samples is probably non-encapsidated and therefore difficult to detect by VIDISCA-NGS.

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