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1.
Medicina (Kaunas) ; 57(12)2021 Dec 09.
Article in English | MEDLINE | ID: covidwho-1572556

ABSTRACT

Background and Objectives: As an mRNA-based vaccine, the Pfizer-BioNTech COVID-19 vaccine has stringent cold storage requirements to preserve functionality of the mRNA active ingredient. To this end, lipid components of the vaccine formulation play an important role in stabilizing and protecting the mRNA molecule for long-term storage. The purpose of the current study was to measure molecular-level dynamics as a function of temperature in the Pfizer-BioNTech COVID-19 vaccine to gain microscopic insight into its thermal stability. Materials and Methods: We used quasielastic and inelastic neutron scattering to probe (1) the vaccine extracted from the manufacturer-supplied vials and (2) unperturbed vaccine in the original manufacturer-supplied vials. The latter measurement was possible due to the high penetrative power of neutrons. Results: Upon warming from the low-temperature frozen state, the vaccine in its original form exhibits two-step melting, indicative of a two-phase morphology. Once the melting is completed (above 0 °C), vaccine re-freezing cannot restore its original two-phase state. This observation is corroborated by the changes in the molecular vibrational spectra. The molecular-level mobility measured in the resulting single-phase state of the re-frozen vaccine greatly exceeds the mobility measured in the original vaccine. Conclusions: Even a brief melting (above 0 °C) leads to an irreversible alteration of the two-phase morphology of the original vaccine formulation. Re-freezing of the vaccine results in a one-phase morphology with much increased molecular-level mobility compared to that in the original vaccine, suggesting irreversible deterioration of the vaccine's in-storage stability. Neutron scattering can be used to distinguish between the vibrational spectra characteristic of the original and deteriorated vaccines contained in the unperturbed original manufacturer-supplied vials.


Subject(s)
COVID-19 , COVID-19 Vaccines , Freezing , Humans , SARS-CoV-2
2.
Diagn Microbiol Infect Dis ; 101(4): 115521, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1363964

ABSTRACT

SARS-CoV-2 whole genome sequencing is a molecular biology tool performed to support many aspects of the response to the pandemic. Freezing of primary clinical nasopharyngeal swabs and shipment to reference laboratories is usually required for sequencing. Cobas PCR Media transport medium facilitates high throughput SARS-CoV-2 RT-PCR analyses on cobas platforms. The manufacturer doesn't recommend freezing this transport medium because of risks of degrading molecular templates and impairing test results. Our objective was to compare the quality and results of SARS-CoV-2 genomic sequencing when performed on fresh or frozen samples in cobas PCR Media. Viral genome sequencing was performed using Oxford Nanopore Technologies MinION platform. Sequencing performance, quality and results did not significantly differ between fresh and frozen samples (n = 10). Freezing of cobas PCR Media does not negatively affect SARS-CoV-2 RNA sequencing results and it is therefore a suitable transport medium for outsourcing sequencing analyses to reference laboratories.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , Freezing , Polymerase Chain Reaction/methods , SARS-CoV-2/isolation & purification , Whole Genome Sequencing/methods , COVID-19/virology , Cryopreservation , Genome, Viral , Humans , Molecular Diagnostic Techniques/methods , Nasopharynx/virology , RNA, Viral/genetics , SARS-CoV-2/genetics
3.
Molecules ; 26(13)2021 Jun 22.
Article in English | MEDLINE | ID: covidwho-1288957

ABSTRACT

In the current work, a simple, economical, accurate, and precise HPLC method with UV detection was developed to quantify Favipiravir (FVIR) in spiked human plasma using acyclovir (ACVR) as an internal standard in the COVID-19 pandemic time. Both FVIR and ACVR were well separated and resolved on the C18 column using the mobile phase blend of methanol:acetonitrile:20 mM phosphate buffer (pH 3.1) in an isocratic mode flow rate of 1 mL/min with a proportion of 30:10:60 %, v/v/v. The detector wavelength was set at 242 nm. Maximum recovery of FVIR and ACVR from plasma was obtained with dichloromethane (DCM) as extracting solvent. The calibration curve was found to be linear in the range of 3.1-60.0 µg/mL with regression coefficient (r2) = 0.9976. However, with acceptable r2, the calibration data's heteroscedasticity was observed, which was further reduced using weighted linear regression with weighting factor 1/x. Finally, the method was validated concerning sensitivity, accuracy (Inter and Intraday's % RE and RSD were 0.28, 0.65 and 1.00, 0.12 respectively), precision, recovery (89.99%, 89.09%, and 90.81% for LQC, MQC, and HQC, respectively), stability (% RSD for 30-day were 3.04 and 1.71 for LQC and HQC, respectively at -20 °C), and carry-over US-FDA guidance for Bioanalytical Method Validation for researchers in the COVID-19 pandemic crisis. Furthermore, there was no significant difference for selectivity when evaluated at LLOQ concentration of 3 µg/mL of FVIR and relative to the blank.


Subject(s)
Amides/analysis , Amides/blood , Antiviral Agents/analysis , Antiviral Agents/blood , Biological Assay/methods , COVID-19/drug therapy , Chromatography, High Pressure Liquid/methods , Liquid-Liquid Extraction/methods , Pyrazines/analysis , Pyrazines/blood , Acyclovir/analysis , Acyclovir/blood , COVID-19/blood , Calibration , Drug Stability , Freezing , Humans , Reference Standards , Reproducibility of Results , Solvents/chemistry
4.
Int J Pharm ; 603: 120701, 2021 Jun 15.
Article in English | MEDLINE | ID: covidwho-1225261

ABSTRACT

In this work, we have developed and tested a dry powder form of niclosamide made by thin-film freezing (TFF) and administered it by inhalation to rats and hamsters to gather data about its toxicology and pharmacokinetics. Niclosamide, a poorly water-soluble drug, is an interesting drug candidate because it was approved over 60 years ago for use as an anthelmintic medication, but recent studies demonstrated its potential as a broad-spectrum antiviral with pharmacological effect against SARS-CoV-2 infection. TFF was used to develop a niclosamide inhalation powder composition that exhibited acceptable aerosol performance with a fine particle fraction (FPF) of 86.0% and a mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of 1.11 µm and 2.84, respectively. This formulation not only proved to be safe after an acute three-day, multi-dose tolerability and exposure study in rats as evidenced by histopathology analysis, and also was able to achieve lung concentrations above the required IC90 levels for at least 24 h after a single administration in a Syrian hamster model. To conclude, we successfully developed a niclosamide dry powder inhalation that overcomes niclosamide's limitation of poor oral bioavailability by targeting the drug directly to the primary site of infection, the lungs.


Subject(s)
COVID-19 , Niclosamide , Administration, Inhalation , Aerosols , Animals , Cricetinae , Dry Powder Inhalers , Freezing , Humans , Particle Size , Powders , Rats , SARS-CoV-2
5.
J Assist Reprod Genet ; 38(4): 783-784, 2021 04.
Article in English | MEDLINE | ID: covidwho-1208603
6.
J Mol Diagn ; 23(6): 691-697, 2021 06.
Article in English | MEDLINE | ID: covidwho-1179822

ABSTRACT

Reliable transportation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) patient samples from a swabbing station to a diagnostics facility is essential for accurate results. Therefore, cooling or freezing the samples is recommended in case of longer transportation times. In this study, SARS-CoV-2 detectability by RT-PCR was assessed after prolonged unfrozen storage or repetitive freeze-thawing of SARS-CoV-2 samples. SARS-CoV-2-positive patient swabs stored in viral transport medium were exposed to different temperatures (4°C, 25°C, and 35°C) and to repetitive freeze-thawing, to assess the effect of storage conditions on RT-PCR detection. SARS-CoV-2 RNA was still reliably detected by RT-PCR after 21 days of storage in viral transport medium, even when the samples had been stored at 35°C. The maximum observed change in cycle threshold value per day was 0.046 (±0.019) at 35°C, and the maximum observed change in cycle threshold value per freeze-thaw cycle per day was 0.197 (±0.06). Compared with storage at 4°C, viral RNA levels deviated little but significantly when stored at 25°C or 35°C, or after repeated freeze-thawing. The results of this study indicate that viral RNA levels are relatively stable at higher temperatures and repetitive freeze-thawing.


Subject(s)
COVID-19 Nucleic Acid Testing/standards , COVID-19/diagnosis , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/standards , SARS-CoV-2/genetics , Specimen Handling/methods , COVID-19/epidemiology , COVID-19 Nucleic Acid Testing/instrumentation , COVID-19 Nucleic Acid Testing/methods , Freezing , Humans , Nasopharynx/virology , RNA Stability , Switzerland/epidemiology , Temperature , Time Factors
7.
J Mol Diagn ; 23(6): 691-697, 2021 06.
Article in English | MEDLINE | ID: covidwho-1155539

ABSTRACT

Reliable transportation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) patient samples from a swabbing station to a diagnostics facility is essential for accurate results. Therefore, cooling or freezing the samples is recommended in case of longer transportation times. In this study, SARS-CoV-2 detectability by RT-PCR was assessed after prolonged unfrozen storage or repetitive freeze-thawing of SARS-CoV-2 samples. SARS-CoV-2-positive patient swabs stored in viral transport medium were exposed to different temperatures (4°C, 25°C, and 35°C) and to repetitive freeze-thawing, to assess the effect of storage conditions on RT-PCR detection. SARS-CoV-2 RNA was still reliably detected by RT-PCR after 21 days of storage in viral transport medium, even when the samples had been stored at 35°C. The maximum observed change in cycle threshold value per day was 0.046 (±0.019) at 35°C, and the maximum observed change in cycle threshold value per freeze-thaw cycle per day was 0.197 (±0.06). Compared with storage at 4°C, viral RNA levels deviated little but significantly when stored at 25°C or 35°C, or after repeated freeze-thawing. The results of this study indicate that viral RNA levels are relatively stable at higher temperatures and repetitive freeze-thawing.


Subject(s)
COVID-19 Nucleic Acid Testing/standards , COVID-19/diagnosis , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/standards , SARS-CoV-2/genetics , Specimen Handling/methods , COVID-19/epidemiology , COVID-19 Nucleic Acid Testing/instrumentation , COVID-19 Nucleic Acid Testing/methods , Freezing , Humans , Nasopharynx/virology , RNA Stability , Switzerland/epidemiology , Temperature , Time Factors
8.
Biotechnol Bioeng ; 118(6): 2202-2219, 2021 06.
Article in English | MEDLINE | ID: covidwho-1098874

ABSTRACT

Serological assays are valuable tools to study SARS-CoV-2 spread and, importantly, to identify individuals that were already infected and would be potentially immune to a virus reinfection. SARS-CoV-2 Spike protein and its receptor binding domain (RBD) are the antigens with higher potential to develop SARS-CoV-2 serological assays. Moreover, structural studies of these antigens are key to understand the molecular basis for Spike interaction with angiotensin converting enzyme 2 receptor, hopefully enabling the development of COVID-19 therapeutics. Thus, it is urgent that significant amounts of this protein became available at the highest quality. In this study, we produced Spike and RBD in two human derived cell hosts: HEK293-E6 and Expi293F™. We evaluated the impact of different and scalable bioprocessing approaches on Spike and RBD production yields and, more importantly, on these antigens' quality attributes. Using negative and positive sera collected from human donors, we show an excellent performance of the produced antigens, assessed in serologic enzyme-linked immunosorbent assay (ELISA) tests, as denoted by the high specificity and sensitivity of the test. We show robust Spike productions with final yields of approx. 2 mg/L of culture that were maintained independently of the production scale or cell culture strategy. To the best of our knowledge, the final yield of 90 mg/L of culture obtained for RBD production, was the highest reported to date. An in-depth characterization of SARS-CoV-2 Spike and RBD proteins was performed, namely the antigen's oligomeric state, glycosylation profiles, and thermal stability during storage. The correlation of these quality attributes with ELISA performance show equivalent reactivity to SARS-CoV-2 positive serum, for all Spike and RBD produced, and for all storage conditions tested. Overall, we provide straightforward protocols to produce high-quality SARS-CoV-2 Spike and RBD antigens, that can be easily adapted to both academic and industrial settings; and integrate, for the first time, studies on the impact of bioprocess with an in-depth characterization of these proteins, correlating antigen's glycosylation and biophysical attributes to performance of COVID-19 serologic tests.


Subject(s)
Antigens, Viral/biosynthesis , Glycosylation , Spike Glycoprotein, Coronavirus/biosynthesis , Cold Temperature , Enzyme-Linked Immunosorbent Assay/standards , Freezing , HEK293 Cells , Humans , Protein Conformation , Protein Stability , Recombinant Proteins/biosynthesis , Recombinant Proteins/standards , SARS-CoV-2 , Serologic Tests/standards , Spike Glycoprotein, Coronavirus/standards
9.
Food Chem ; 349: 129178, 2021 Jul 01.
Article in English | MEDLINE | ID: covidwho-1065087

ABSTRACT

Chinese steamed breads (CSB) and noodles are staple foods for many people. The production of frozen steamed products and boiled noodles has kept increasing. This is due to the increasing demand of ready-to-eat frozen food products from the market. Frozen storage significantly increases the self-life of the products and reduces the production costs. On the other hand, the freezing and frozen storage lead to quality loss of the frozen products. This review summarizes effects of freezing and frozen storage on diverse quality attributes (e.g., structural and textural properties) of frozen northern-type steamed breads and boiled noodles. Food safety of the frozen products related to the COVID-19 pandemic is discussed. To counteract the quality loss of the frozen products, suitable processing methods, selection of basic ingredients and uses of various food additives can be done. Research gaps to improve the textural, cooking and nutritional quality of frozen CSB and noodles are suggested.


Subject(s)
Bread/analysis , Flour/analysis , Food Storage , Cooking , Freezing , Humans , Nutritive Value , Steam
10.
J Clin Virol ; 133: 104686, 2020 12.
Article in English | MEDLINE | ID: covidwho-1014611

ABSTRACT

INTRODUCTION: Studies describing the performance characteristics of the cobas®6800 system for SARS-CoV-2 detection in deep respiratory specimens and freeze-thaw stability are limited. The current study compares the clinical performance of the automated SARS-CoV-2 assay on the cobas®6800 system to a lab-developed assay (LDA) and the cobas impact of freeze-thawing combined with lysis buffer. METHODS: Both retrospective and prospectively selected deep respiratory samples and oro- and nasopharyngeal samples in either E-swab® or GLY- were tested using the SARS-CoV-2 assay on the cobas®6800 System and compared to a lab developed assay. Additonally, SARS-CoV-2 RNA stability was assessed after one freeze-thaw cycle with or without lysis buffer. RESULTS: In total, 221 (58.3 %) oro- and nasopharyngeal swabs, 131 (34.6 %) deep respiratory specimens, and n = 25 (6.6 %) swabs of unknown origin were included to study clinical performance. Only 4 samples gave discrepant results, all being positive in the LDA and not the cobas®6800 system. For stability testing, 66 samples without and 110 with lysis buffer were included. No clinically significant difference was found in test results after one freeze-thaw cycle and addition of lysis buffer. CONCLUSION: Based on our findings, the cobas®6800 SARS-CoV-2 RNA assay yielded similar results as the LDA in oro-/nasopharyngeal swabs and deep respiratory specimens. Moreover, the cobas®6800 SARS-CoV-2 RNA assay yielded similar results before and after a freeze-thaw cycle, with better preservation of low viral loads in lysis buffer.


Subject(s)
COVID-19 Nucleic Acid Testing , COVID-19/diagnosis , Freezing , Nasopharynx/virology , Respiratory System/virology , Specimen Handling/methods , Feces/virology , Humans , Prospective Studies , RNA, Viral/genetics , Reagent Kits, Diagnostic , Retrospective Studies , SARS-CoV-2/genetics , Viral Load
12.
Angew Chem Int Ed Engl ; 60(1): 321-330, 2021 01 04.
Article in English | MEDLINE | ID: covidwho-891871

ABSTRACT

Matching of symmetry at interfaces is a fundamental obstacle in molecular assembly. Virus-like particles (VLPs) are important vaccine platforms against pathogenic threats, including Covid-19. However, symmetry mismatch can prohibit vaccine nanoassembly. We established an approach for coupling VLPs to diverse antigen symmetries. SpyCatcher003 enabled efficient VLP conjugation and extreme thermal resilience. Many people had pre-existing antibodies to SpyTag:SpyCatcher but less to the 003 variants. We coupled the computer-designed VLP not only to monomers (SARS-CoV-2) but also to cyclic dimers (Newcastle disease, Lyme disease), trimers (influenza hemagglutinins), and tetramers (influenza neuraminidases). Even an antigen with dihedral symmetry could be displayed. For the global challenge of influenza, SpyTag-mediated display of trimer and tetramer antigens strongly induced neutralizing antibodies. SpyCatcher003 conjugation enables nanodisplay of diverse symmetries towards generation of potent vaccines.


Subject(s)
COVID-19 Vaccines/chemistry , Nanostructures/chemistry , Vaccines, Virus-Like Particle/chemistry , Antibodies, Neutralizing/analysis , Antibodies, Viral , Antigens, Viral/chemistry , Antigens, Viral/immunology , Freezing , Humans , Models, Molecular
13.
J Infect Dis ; 222(2): 203-205, 2020 06 29.
Article in English | MEDLINE | ID: covidwho-306065

ABSTRACT

The detection of SARS-CoV-2 infection is the premise of quarantine. In many countries or areas, samples need to be shipped or inactivated before SARS-CoV-2 testing. In this study, we checked the influence of sample storage conditions on SARS-CoV-2 nucleic acid testing results, including sample inactivation time, storage temperature, and storage time. All of these conditions caused an increase in the cycle threshold values of the nucleic acid tests and led to the misclassification of at least 10.2% of positive cases as negative or suspected. The results highlight the importance of immediate testing of samples for SARS-CoV-2 nucleic acid detection.


Subject(s)
Betacoronavirus , Clinical Laboratory Techniques , Coronavirus Infections/diagnosis , Pharynx/virology , Pneumonia, Viral/diagnosis , Specimen Handling/methods , Betacoronavirus/genetics , COVID-19 , COVID-19 Testing , Cryopreservation , Freezing , Humans , Pandemics , Refrigeration , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2 , Temperature , Time Factors , Virus Inactivation
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