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1.
PubMed; 2021.
Preprint in English | PubMed | ID: ppcovidwho-333666

ABSTRACT

The exact mechanism of coronavirus replication and transcription is not fully understood;however, a hallmark of coronavirus transcription is the generation of negative-sense RNA intermediates that serve as the templates for the synthesis of positive-sense genomic RNA (gRNA) and an array of subgenomic mRNAs (sgRNAs) encompassing sequences arising from discontinuous transcription. Existing PCR-based diagnostic assays for SAR-CoV-2 are qualitative or semi-quantitative and do not provide the resolution needed to assess the complex transcription dynamics of SARS-CoV-2 over the course of infection. We developed and validated a novel panel of specially designed SARS-CoV-2 ddPCR-based assays to map the viral transcription profile. Application of these assays to clinically relevant samples will enhance our understanding of SARS-CoV-2 replication and transcription and may also inform the development of improved diagnostic tools and therapeutics. HIGHLIGHTS: We developed a novel panel of 7 quantitative RT-ddPCRs assays for SARS-Cov-2Our panel targets nongenic and genic regions in genomic and subgenomic RNAsAll assays detect 1-10 copies and are linear over 3-4 orders of magnitudeAll assays correlated with the clinical Abbott SARS-CoV-2 Viral Load AssayClinical samples showed higher copy numbers for targets at the 3' end of the genome.

2.
PLoS ONE ; 16(2), 2021.
Article in English | CAB Abstracts | ID: covidwho-1410710

ABSTRACT

Background: Sensitive and high throughput molecular detection assays are essential during the coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The vast majority of the SARS-CoV-2 molecular assays use nasopharyngeal swab (NPS) or oropharyngeal swab (OPS) specimens collected from suspected individuals. However, using NPS or OPS as specimens has apparent drawbacks, e.g. the collection procedures for NPS or OPS specimens can be uncomfortable to some people and may cause sneezing and coughing which in turn generate droplets and/or aerosol particles that are of risk to healthcare workers, requiring heavy use of personal protective equipment. There have been recent studies indicating that self-collected saliva specimens can be used for molecular detection of SARS-CoV-2 and provides more comfort and ease of use for the patients. Here we report the performance of QuantiVirusTM SARS-CoV-2 test using saliva as the testing specimens with or without pooling. Methods Development and validation studies were conducted following FDA-EUA and molecular assay validation guidelines. Using SeraCare Accuplex SARS-CoV-2 reference panel, the limit of detection (LOD) and clinical performance studies were performed with the QuantiVirusTM SARS-CoV-2 test. For clinical evaluation, 85 known positive and 90 known negative clinical NPS samples were tested. Additionally, twenty paired NPS and saliva samples collected from recovering COVID-19 patients were tested and the results were further compared to that of the Abbott m2000 SARS-CoV-2 PCR assay. Results of community collected 389 saliva samples for COVID-19 screening by QuantiVirusTM SARS-CoV-2 test were also obtained and analyzed. Additionally, testing of pooled saliva samples was evaluated.

3.
Topics in Antiviral Medicine ; 29(1):62, 2021.
Article in English | EMBASE | ID: covidwho-1250779

ABSTRACT

Background: SARS-CoV-2 is transcribed as genomic RNA (gRNA) and different subgenomic RNAs (sgRNA), allowing variation in viral gene expression. However, the extent and clinical significance of subgenomic transcription remain unknown. We hypothesized that SARS-CoV-2 RNA levels would vary between genome regions and between patients, tissues, and sample types (cells or fluids). Methods: We designed and validated 7 novel RT-ddPCR assays that target the 5' and 3' untranslated regions (UTR), non-structural genes found only in full length gRNA [Main Proteinase (NSP5) and RNA dependent RNA polymerase (RdRp)], and 3' structural genes [Spike (S), membrane (M), and nucleocapsid (N)] that are also contained in different sgRNAs. Assay efficiencies were measured on standards derived from plasmids and viral stock supernatants. Levels of all 7 RNA regions were measured in nucleic acid extracted by the Abbott m2000 platform from nasopharyngeal (NP) swabs from 3 SARS-CoV-2 infected individuals, and in cells and supernatant from NP, oropharynx (OP), and saliva isolated from 3 additional individuals. Results: In all samples, levels of 3' targets (M, N, and 3'UTR) tended to be higher than 5' targets (5' UTR, NSP5, and RdRp), suggesting an excess of 3' sgRNAs (3'UTR/5'UTR=2.4-6.2 and nucleocapsid/RdRp=1.1-7.5 for NP samples;n=6, p=0.03). All SARS-CoV-2 RNAs were detected in both cells and supernatant from NP, OP, and saliva, but tended to be higher in the NP than OP. In saliva but not NP or OP, levels of gRNA/μL sample were consistently higher in the cells compared to supernatant (cell/sup=2.7-44.8). Surprisingly, the excess of 3' over 5' viral RNAs was even greater in the supernatant (3'UTR/5'UTR=8.2-38.7) compared to cells (3'UTR/5'UTR=1.5-6.2) from NP, OP, and saliva (p=0.016 across all), suggesting a greater excess of sgRNAs in the cell-free fluids. Conclusion: The higher levels of 3' targets suggest an excess of sgRNA in all samples. Assays that target 3' regions found in sgRNAs (N, 3'UTR) may be more sensitive for detecting SARS-CoV-2, but may not indicate infectious virus. The greater excess of 3' transcripts in cell-free fluids suggests that sgRNAs are released from cells and/or persist to a greater degree than gRNAs. Future studies should investigate how levels of sgRNA change over the course of infection in cells and cell-free fluids, and whether sgRNA levels correlate with measures of disease transmission or severity.

5.
Anaesthesia ; 75(8): 1022-1027, 2020 08.
Article in English | MEDLINE | ID: covidwho-751832

ABSTRACT

The COVID-19 pandemic has increased the demand for disposable N95 respirators. Re-usable elastomeric respirators may provide a suitable alternative. Proprietary elastomeric respirator filters may become depleted as demand increases. An alternative may be the virus/bacterial filters used in anaesthesia circuits, if they can be adequately fitted onto the elastomeric respirators. In addition, many re-usable elastomeric respirators do not filter exhaled breaths. If used for sterile procedures, this would also require modification. We designed a 3D-printed adaptor that permits elastomeric respirators to interface with anaesthesia circuit filters and created a simple modification to divert exhaled breaths through the filter. We conducted a feasibility study evaluating the performance of our modified elastomeric respirators. A convenience sample of eight volunteers was recruited. Quantitative fit testing, respiratory rate and end-tidal carbon dioxide were recorded during fit testing exercises and after 1 h of wear. All eight volunteers obtained excellent quantitative fit testing throughout the trial. The mean (SD) end-tidal carbon dioxide was 4.5 (0.5) kPa and 4.6 (0.4) kPa at baseline and after 1 h of wear (p = 0.148). The mean (SD) respiratory rate was 17 (4) breaths.min-1 and 17 (3) breaths.min-1 at baseline and after 1 h of wear (p = 0.435). Four out of eight subjects self-reported discomfort; two reported facial pressure, one reported exhalation resistance and one reported transient dizziness on exertion. Re-usable elastomeric respirators to utilise anaesthesia circuit filters through a 3D-printed adaptor may be a potential alternative to disposable N95 respirators during the COVID-19 pandemic.


Subject(s)
Betacoronavirus , Coronavirus Infections/therapy , Filtration/instrumentation , Pneumonia, Viral/therapy , Ventilators, Mechanical , Adult , COVID-19 , Carbon Dioxide/physiology , Coronavirus Infections/epidemiology , Elastomers , Equipment Design , Equipment Reuse , Feasibility Studies , Female , Humans , Male , Materials Testing/methods , Middle Aged , Pandemics , Pneumonia, Viral/epidemiology , Printing, Three-Dimensional , Respiratory Rate , SARS-CoV-2 , Ventilators, Mechanical/supply & distribution
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