scholarly journals Heat Inactivation of Different Types of SARS-CoV-2 Samples: What Protocols for Biosafety, Molecular Detection and Serological Diagnostics?

Viruses ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 735 ◽  
Author(s):  
Boris Pastorino ◽  
Franck Touret ◽  
Magali Gilles ◽  
Xavier de Lamballerie ◽  
Remi N. Charrel
Keyword(s):  
Infected Cell ◽  
Limit Of Detection ◽  
Elisa Test ◽  
Cell Culture Supernatant ◽  
Heat Inactivation ◽  
Standard Precautions ◽  
Viral Loads ◽  
Human Sera ◽  
European Norm ◽  
C 30

Standard precautions to minimize the risk of SARS-CoV-2 transmission implies that infected cell cultures and clinical specimens may undergo some sort of inactivation to reduce or abolish infectivity. We evaluated three heat inactivation protocols (56 °C-30 min, 60 °C-60 min and 92 °C-15 min) on SARS-CoV-2 using (i) infected cell culture supernatant, (ii) virus-spiked human sera (iii) and nasopharyngeal samples according to the recommendations of the European norm NF EN 14476-A2. Regardless of the protocol and the type of samples, a 4 Log10 TCID50 reduction was observed. However, samples containing viral loads > 6 Log10 TCID50 were still infectious after 56 °C-30 min and 60 °C-60 min, although infectivity was < 10 TCID50. The protocols 56 °C-30 min and 60 °C-60 min had little influence on the RNA copies detection, whereas 92 °C-15 min drastically reduced the limit of detection, which suggests that this protocol should be avoided for inactivation ahead of molecular diagnostics. Lastly, 56 °C-30 min treatment of serum specimens had a negligible influence on the results of IgG detection using a commercial ELISA test, whereas a drastic decrease in neutralizing titers was observed.

An RNA polymerase II transcription factor inactivated in poliovirus-infected cells copurifies with transcription factor TFIID

1988 ◽  
Vol 8 (8) ◽  
pp. 3175-3182
Author(s):  
S Kliewer ◽  
A Dasgupta
Keyword(s):  
Transcription Factor ◽  
Infected Cell ◽  
Rna Polymerase ◽  
Host Cell ◽  
Rna Polymerase Ii ◽  
Heat Inactivation ◽  
Early Event ◽  
Cell Extracts ◽  
Infected Cells ◽  
Rna Polymerase Ii Transcription

Inhibition of host cell RNA polymerase II-mediated transcription by poliovirus infection was studied in vitro. Whole-cell extracts prepared from poliovirus-infected HeLa cells at 3 h postinfection were shown to be deficient in a factor required for specific transcription from the adenovirus major late promoter. Three lines of evidence suggest that transcription factor TFIID is deficient in poliovirus-infected cells. First, the activity required to specifically restore transcription in poliovirus-infected cell extracts was shown to copurify with TFIID through three chromatographic steps. Second, transcription reactions reconstituted with phosphocellulose-derived chromatographic fractions revealed a fourfold decrease in the specific activity of the TFIID-containing fraction prepared from poliovirus-infected cells compared with that of the same fraction prepared from mock-infected cells. Finally, TFIID and the activity required to specifically restore transcription in virus-infected cell extracts were shown to have the same kinetics of heat inactivation. Together, these results suggest that inactivation of TFIID is an early event in the inhibition of host cell RNA polymerase II transcription by poliovirus.

scholarly journals The impact of thermal pasteurization on viral load and detectable live viruses in human milk and other matrices: a rapid review

2021 ◽  
Vol 46 (1) ◽  
pp. 10-26 ◽  
Author(s):  
Michael A. Pitino ◽  
Deborah L. O’Connor ◽  
Allison J. McGeer ◽  
Sharon Unger
Keyword(s):  
Viral Load ◽  
Human Milk ◽  
Limit Of Detection ◽  
Culture Media ◽  
Rapid Review ◽  
Future Research ◽  
Live Virus ◽  
C 30 ◽  
The Impact ◽  
Thermal Pasteurization

Holder pasteurization (62.5 °C, 30 min) of human milk is thought to reduce the risk of transmitting viruses to an infant. Some viruses may be secreted into milk – others may be contaminants. The effect of thermal pasteurization on viruses in human milk has yet to be rigorously reviewed. The objective of this study is to characterize the effect of common pasteurization techniques on viruses in human milk and non-human milk matrices. Databases (MEDLINE, Embase, Web of Science) were searched from inception to April 20th, 2020, for primary research articles assessing the impact of pasteurization on viral load or detection of live virus. Reviews were excluded, as were studies lacking quantitative measurements or those assessing pasteurization as a component of a larger process. Overall, of 65 131 reports identified, 109 studies were included. Pasteurization of human milk at a minimum temperature of 56−60 °C is effective at reducing detectable live virus. In cell culture media or plasma, coronaviruses (e.g., SARS-CoV, SARS-CoV-2, MERS-CoV) are highly susceptible to heating at ≥56 °C. Although pasteurization parameters and matrices reported vary, all viruses studied, except parvoviruses, were susceptible to thermal killing. Future research important for the study of novel viruses should standardize pasteurization protocols and should test inactivation in human milk. Novelty In all matrices, including human milk, pasteurization at 62.5 °C was generally sufficient to reduce surviving viral load by several logs or to below the limit of detection. Holder pasteurization (62.5 °C, 30 min) of human milk should be sufficient to inactivate nonheat resistant viruses, including coronaviruses, if present.

scholarly journals The effect of temperature on persistence of SARS-CoV-2 on common surfaces

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Shane Riddell ◽  
Sarah Goldie ◽  
Andrew Hill ◽  
Debbie Eagles ◽  
Trevor W. Drew
Keyword(s):  
Uv Light ◽  
Risk Mitigation ◽  
Survival Rates ◽  
Environmental Stability ◽  
Effect Of Temperature ◽  
Viral Loads ◽  
Different Temperatures ◽  
C 30 ◽  
D Values

Abstract Background The rate at which COVID-19 has spread throughout the globe has been alarming. While the role of fomite transmission is not yet fully understood, precise data on the environmental stability of SARS-CoV-2 is required to determine the risks of fomite transmission from contaminated surfaces. Methods This study measured the survival rates of infectious SARS-CoV-2, suspended in a standard ASTM E2197 matrix, on several common surface types. All experiments were carried out in the dark, to negate any effects of UV light. Inoculated surfaces were incubated at 20 °C, 30 °C and 40 °C and sampled at various time points. Results Survival rates of SARS-CoV-2 were determined at different temperatures and D-values, Z-values and half-life were calculated. We obtained half lives of between 1.7 and 2.7 days at 20 °C, reducing to a few hours when temperature was elevated to 40 °C. With initial viral loads broadly equivalent to the highest titres excreted by infectious patients, viable virus was isolated for up to 28 days at 20 °C from common surfaces such as glass, stainless steel and both paper and polymer banknotes. Conversely, infectious virus survived less than 24 h at 40 °C on some surfaces. Conclusion These findings demonstrate SARS-CoV-2 can remain infectious for significantly longer time periods than generally considered possible. These results could be used to inform improved risk mitigation procedures to prevent the fomite spread of COVID-19.

scholarly journals Microfluidic Nano-Scale qPCR Enables Ultra-Sensitive and Quantitative Detection of SARS-CoV-2

Processes ◽  
2020 ◽  
Vol 8 (11) ◽  
pp. 1425
Author(s):  
Xin Xie ◽  
Tamara Gjorgjieva ◽  
Zaynoun Attieh ◽  
Mame Massar Dieng ◽  
Marc Arnoux ◽  
...  
Keyword(s):  
Viral Infections ◽  
Limit Of Detection ◽  
False Negative ◽  
False Negative Rate ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
Rt Pcr ◽  
Viral Loads ◽  
Nano Scale ◽  
Negative Rate

A major challenge in controlling the COVID-19 pandemic is the high false-negative rate of the commonly used RT-PCR methods for SARS-CoV-2 detection in clinical samples. Accurate detection is particularly challenging in samples with low viral loads that are below the limit of detection (LoD) of standard one- or two-step RT-PCR methods. In this study, we implemented a three-step approach for SARS-CoV-2 detection and quantification that employs reverse transcription, targeted cDNA preamplification, and nano-scale qPCR based on a commercially available microfluidic chip. Using SARS-CoV-2 synthetic RNA and plasmid controls, we demonstrate that the addition of a preamplification step enhances the LoD of this microfluidic RT-qPCR by 1000-fold, enabling detection below 1 copy/µL. We applied this method to analyze 182 clinical NP swab samples previously diagnosed using a standard RT-qPCR protocol (91 positive, 91 negative) and demonstrate reproducible and quantitative detection of SARS-CoV-2 over five orders of magnitude (<1 to 106 viral copies/µL). Crucially, we detect SARS-CoV-2 with relatively low viral load estimates (<1 to 40 viral copies/µL) in 17 samples with negative clinical diagnosis, indicating a potential false-negative rate of 18.7% by clinical diagnostic procedures. In summary, this three-step nano-scale RT-qPCR method can robustly detect SARS-CoV-2 in samples with relatively low viral loads (<1 viral copy/µL) and has the potential to reduce the false-negative rate of standard RT-PCR-based diagnostic tests for SARS-CoV-2 and other viral infections.

scholarly journals Direct-RT-qPCR Detection of SARS-CoV-2 without RNA Extraction as Part of a COVID-19 Testing Strategy: From Sample to Result in One Hour

Diagnostics ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 605 ◽  
Author(s):  
Eva Kriegova ◽  
Regina Fillerova ◽  
Petr Kvapil
Keyword(s):  
High Throughput Screening ◽  
High Speed ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Rna Extraction ◽  
Rna Isolation ◽  
High Sensitivity ◽  
Ease Of Use ◽  
Diagnostic Tools ◽  
Heat Inactivation

Due to the lack of protective immunity in the general population and the absence of effective antivirals and vaccines, the Coronavirus disease 2019 (COVID-19) pandemic continues in some countries, with local epicentres emerging in others. Due to the great demand for effective COVID-19 testing programmes to control the spread of the disease, we have suggested such a testing programme that includes a rapid RT-qPCR approach without RNA extraction. The Direct-One-Step-RT-qPCR (DIOS-RT-qPCR) assay detects severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in less than one hour while maintaining the high sensitivity and specificity required of diagnostic tools. This optimised protocol allows for the direct use of swab transfer media (14 μL) without the need for RNA extraction, achieving comparable sensitivity to the standard method that requires the time-consuming and costly step of RNA isolation. The limit of detection for DIOS-RT-qPCR was lower than seven copies/reaction, which translates to 550 virus copies/mL of swab. The speed, ease of use and low price of this assay make it suitable for high-throughput screening programmes. The use of fast enzymes allows RT-qPCR to be performed under standard laboratory conditions within one hour, making it a potential point-of-care solution on high-speed cycling instruments. This protocol also implements the heat inactivation of SARS-CoV-2 (75 °C for 10 min), which renders samples non-infectious, enabling testing in BSL-2 facilities. Moreover, we discuss the critical steps involved in developing tests for the rapid detection of COVID-19. Implementing rapid, easy, cost-effective methods can help control the worldwide spread of the COVID-19 infection.

scholarly journals Highly Sensitive and Specific SARS-CoV-2 Serological Assay Using a Magnetic Modulation Biosensing System

Biosensors ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 7
Author(s):  
Shira Avivi-Mintz ◽  
Yaniv Lustig ◽  
Victoria Indenbaum ◽  
Eli Schwartz ◽  
Amos Danielli
Keyword(s):  
Viral Infections ◽  
Limit Of Detection ◽  
Turnaround Time ◽  
Elisa Test ◽  
Antibody Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Antibodies ◽  
Serological Assay ◽  
Clinical Sensitivity ◽  
Highly Sensitive

Sensitive serological assays are needed to provide valuable information about acute and past viral infections. For example, detection of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgG antibodies could serve as the basis for an “immunity passport” that would enable individuals to travel internationally. Here, utilizing a novel Magnetic Modulation Biosensing (MMB) system and the receptor-binding domain of the SARS-CoV-2 spike protein, we demonstrate a highly sensitive and specific anti-SARS-CoV-2 IgG serological assay. Using anti-SARS-CoV-2 IgG antibodies, RT-qPCR SARS-CoV-2-positive and healthy patients’ samples, and vaccinees’ samples, we compare the MMB-based SARS-CoV-2 IgG assay’s analytical and clinical sensitivities to those of the enzyme-linked immunosorbent assay (ELISA). Compared with ELISA, the MMB-based assay has an ~6-fold lower limit of detection (129 ng/L vs. 817 ng/L), and it detects an increase in the IgG concentration much earlier after vaccination. Using 85 RT-qPCR SARS-CoV-2-positive samples and 79 -negative samples, the MMB-based assay demonstrated similar clinical specificity (98% vs. 99%) and sensitivity (93% vs. 92%) to the ELISA test, but with a much faster turnaround time (45 min vs. 245 min). The high analytical and clinical sensitivity, short turnaround time, and simplicity of the MMB-based assay makes it a preferred method for antibody detection.

scholarly journals A Saliva-Based RNA Extraction-Free Workflow Integrated With Cas13a for SARS-CoV-2 Detection

2021 ◽  
Vol 11 ◽  
Author(s):  
Iqbal Azmi ◽  
Md Imam Faizan ◽  
Rohit Kumar ◽  
Siddharth Raj Yadav ◽  
Nisha Chaudhary ◽  
...  
Keyword(s):  
Data Storage ◽  
Limit Of Detection ◽  
Rna Extraction ◽  
Smartphone Application ◽  
Lateral Flow ◽  
Clinical Samples ◽  
Heat Inactivation ◽  
Analytical Sensitivity ◽  
Test Results ◽  
Extraction Step

A major bottleneck in scaling-up COVID-19 testing is the need for sophisticated instruments and well-trained healthcare professionals, which are already overwhelmed due to the pandemic. Moreover, the high-sensitive SARS-CoV-2 diagnostics are contingent on an RNA extraction step, which, in turn, is restricted by constraints in the supply chain. Here, we present CASSPIT (Cas13AssistedSaliva-based &amp;SmartphoneIntegratedTesting), which will allow direct use of saliva samples without the need for an extra RNA extraction step for SARS-CoV-2 detection. CASSPIT utilizes CRISPR-Cas13a based SARS-CoV-2 RNA detection, and lateral-flow assay (LFA) readout of the test results. The sample preparation workflow includes an optimized chemical treatment and heat inactivation method, which, when applied to COVID-19 clinical samples, showed a 97% positive agreement with the RNA extraction method. With CASSPIT, LFA based visual limit of detection (LoD) for a given SARS-CoV-2 RNA spiked into the saliva samples was ~200 copies; image analysis-based quantification further improved the analytical sensitivity to ~100 copies. Upon validation of clinical sensitivity on RNA extraction-free saliva samples (n = 76), a 98% agreement between the lateral-flow readout and RT-qPCR data was found (Ct&lt;35). To enable user-friendly test results with provision for data storage and online consultation, we subsequently integrated lateral-flow strips with a smartphone application. We believe CASSPIT will eliminate our reliance on RT-qPCR by providing comparable sensitivity and will be a step toward establishing nucleic acid-based point-of-care (POC) testing for COVID-19.

scholarly journals Evaluation of In-Flow Magnetoresistive Chip Cell—Counter as a Diagnostic Tool

Biosensors ◽  
2019 ◽  
Vol 9 (3) ◽  
pp. 105 ◽  
Author(s):  
Manon Giraud ◽  
François-Damien Delapierre ◽  
Anne Wijkhuisen ◽  
Pierre Bonville ◽  
Mathieu Thévenin ◽  
...  
Keyword(s):  
Giant Magnetoresistance ◽  
Limit Of Detection ◽  
Malignant Cell ◽  
Elisa Test ◽  
Great Sensitivity ◽  
Actual System ◽  
Whole Cells ◽  
Gmr Sensors ◽  
Gmr Sensor ◽  
Sensor Detection

Inexpensive simple medical devices allowing fast and reliable counting of whole cells are of interest for diagnosis and treatment monitoring. Magnetic-based labs on a chip are one of the possibilities currently studied to address this issue. Giant magnetoresistance (GMR) sensors offer both great sensitivity and device integrability with microfluidics and electronics. When used on a dynamic system, GMR-based biochips are able to detect magnetically labeled individual cells. In this article, a rigorous evaluation of the main characteristics of this magnetic medical device (specificity, sensitivity, time of use and variability) are presented and compared to those of both an ELISA test and a conventional flow cytometer, using an eukaryotic malignant cell line model in physiological conditions (NS1 murine cells in phosphate buffer saline). We describe a proof of specificity of a GMR sensor detection of magnetically labeled cells. The limit of detection of the actual system was shown to be similar to the ELISA one and 10 times higher than the cytometer one.

Pulmonary lesions induced by SARS-CoV-2 infection in domestic cats

2021 ◽  
pp. 030098582110668
Author(s):  
Olivia M. Patania ◽  
Shiho Chiba ◽  
Peter J. Halfmann ◽  
Masato Hatta ◽  
Tadashi Maemura ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Acute Infection ◽  
Viral Rna ◽  
Vacuolar Degeneration ◽  
Pulmonary Lesions ◽  
Viral Loads ◽  
Mhc Ii ◽  
Type Ii Pneumocytes ◽  
Microscopic Evaluation

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019, which ranges from fatal disease in some to mild or subclinical in most affected individuals. Many recovered human patients report persistent respiratory signs; however, lung disease in post-acute infection is poorly understood. Our objective was to describe histologic lung lesions and viral loads following experimental SARS-CoV-2 infection in 11 cats. Microscopic evaluation at 3, 6, 10, or 28 days postinoculation (DPI) identified mild to moderate patchy interstitial pneumonia, bronchiolar epithelial damage, and occlusive histiocytic bronchiolitis. Based on immunohistochemistry, alveolar septal thickening was due to CD204-positive macrophages, fewer B and T lymphocytes, type II pneumocytes, and capillary proliferation with a relative dearth of fibrosis. In blood vessel endothelium, there was reactive hypertrophy or vacuolar degeneration and increased MHC II expression at all time points. Unexpectedly, one cat from the 28 DPI group had severe subacute regionally extensive lymphohistiocytic pneumonia with multifocal consolidation, vasculitis, and alveolar fibrin. Reverse transcriptase-quantitative polymerase chain reaction identified SARS-CoV-2 RNA within the lung at 3 and 6 DPI, and viral RNA was below the limit of detection at 10 and 28 DPI, suggesting that pulmonary lesions persist beyond detection of viral RNA. These findings clarify our comparative understanding of disease induced by SARS-CoV-2 and suggest that cats can serve as an informative model to study post-acute pulmonary sequelae.

scholarly journals The SARS-CoV-2 viral load in COVID-19 patients is lower on face mask filters than on nasopharyngeal swabs

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Agnieszka Smolinska ◽  
David S. Jessop ◽  
Kirk L. Pappan ◽  
Alexandra De Saedeleer ◽  
Amerjit Kang ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Face Mask ◽  
Protective Equipment ◽  
Viral Loads ◽  
Non Invasive ◽  
Home Testing ◽  
Viral Particles ◽  
Mean Diameter ◽  
The Face ◽  
Respiratory Droplets

AbstractFace masks and personal respirators are used to curb the transmission of SARS-CoV-2 in respiratory droplets; filters embedded in some personal protective equipment could be used as a non-invasive sample source for applications, including at-home testing, but information is needed about whether filters are suited to capture viral particles for SARS-CoV-2 detection. In this study, we generated inactivated virus-laden aerosols of 0.3–2 microns in diameter (0.9 µm mean diameter by mass) and dispersed the aerosolized viral particles onto electrostatic face mask filters. The limit of detection for inactivated coronaviruses SARS-CoV-2 and HCoV-NL63 extracted from filters was between 10 to 100 copies/filter for both viruses. Testing for SARS-CoV-2, using face mask filters and nasopharyngeal swabs collected from hospitalized COVID-19-patients, showed that filter samples offered reduced sensitivity (8.5% compared to nasopharyngeal swabs). The low concordance of SARS-CoV-2 detection between filters and nasopharyngeal swabs indicated that number of viral particles collected on the face mask filter was below the limit of detection for all patients but those with the highest viral loads. This indicated face masks are unsuitable to replace diagnostic nasopharyngeal swabs in COVID-19 diagnosis. The ability to detect nucleic acids on face mask filters may, however, find other uses worth future investigation.

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