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1.
Genet Test Mol Biomarkers ; 25(2): 85-101, 2021 Feb.
Article in English | MEDLINE | ID: covidwho-1091280

ABSTRACT

Coronavirus disease 2019 (COVID-19) displays a broad spectrum of clinical presentations ranging from lack of symptoms to severe multiorgan system complications and death. Various laboratory assays have been employed in the diagnosis of COVID-19, including: nucleic acid-based tests; antigen tests; and serum testing for anti-severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antibodies. The disease can also be diagnosed based on suggestive clinical features and radiological findings. Until now, remdesivir is the only medication approved for the treatment of COVID-19 by the U.S. Food and Drug Administration (FDA); however, it is anticipated that several anti-SARS-CoV-2 monoclonal antibodies will gain soon approval. Other methods of treatment include supportive care directed toward treating the symptoms. Nevertheless, many studies have recently emerged, showing controversial preliminary results with the off-label medication hydroxychloroquine. Given that all results are still preliminary, including those seen by remdesivir, additional evidence and research are required to identify effective medications that are broadly effective and well tolerated. Importantly, two RNA-based vaccines have recently gained approval from Pfizer and Moderna, with many others still in clinical trials. This article reviews various aspects of COVID-19, including its epidemiology; its evolution and mutational spectrum; and its clinical dynamics, symptoms and complications, diagnosis, and treatment.


Subject(s)
Global Burden of Disease/statistics & numerical data , Pandemics/statistics & numerical data , /pathogenicity , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/therapeutic use , Alanine/analogs & derivatives , Alanine/therapeutic use , Antiviral Agents/therapeutic use , /drug therapy , /therapy , /methods , Clinical Trials as Topic , Evolution, Molecular , Humans , Hydroxychloroquine/therapeutic use , Mutation , Off-Label Use , Pandemics/prevention & control , RNA, Viral/genetics , RNA, Viral/isolation & purification , /immunology , Severity of Illness Index
2.
J Transl Med ; 19(1): 74, 2021 02 16.
Article in English | MEDLINE | ID: covidwho-1088600

ABSTRACT

BACKGROUND: COVID-19 has spread rapidly around the world, affecting a large percentage of the population. When lifting certain mandatory measures for an economic restart, robust surveillance must be established and implemented, with nucleic acid detection for SARS-CoV-2 as an essential component. METHODS: We tried to develop a one-tube detection platform based on RT-RPA (Reverse Transcription and Recombinase Polymerase Isothermal Amplification) and DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR) technology, termed OR-DETECTR, to detect SARS-CoV-2. We designed RT-RPA primers of the RdRp and N genes following the SARS-CoV-2 gene sequence. We optimized reaction components so that the detection process could be carried out in one tube. Specificity was demonstrated by detecting nucleic acid samples from pseudoviruses from seven human coronaviruses and Influenza A (H1N1). Clinical samples were used to validate the platform and all results were compared to rRT-PCR. RNA standards and pseudoviruses diluted by different gradients were used to demonstrate the detection limit. Additionally, we have developed a lateral flow assay based on OR-DETECTR for detecting COVID-19. RESULTS: The OR-DETECTR detection process can be completed in one tube, which takes approximately 50 min. This method can specifically detect SARS-CoV-2 from seven human coronaviruses and Influenza A (H1N1), with a low detection limit of 2.5 copies/µl input (RNA standard) and 1 copy/µl input (pseudovirus). Results of six samples from SARS-CoV-2 patients, eight samples from patients with fever but no SARS-CoV-2 infection, and one mixed sample from 40 negative controls showed that OR-DETECTR is 100% consistent with rRT-PCR. The lateral flow assay based on OR-DETECTR can be used for the detection of COVID-19, and the detection limit is 2.5 copies/µl input. CONCLUSIONS: The OR-DETECTR platform for the detection of COVID-19 is rapid, accurate, tube closed, easy-to-operate, and free of large instruments.


Subject(s)
/methods , /virology , CRISPR-Cas Systems/genetics , Real-Time Polymerase Chain Reaction/methods , Reverse Transcription/genetics , /isolation & purification , Base Sequence , Humans , Limit of Detection , RNA, Viral/genetics , Reference Standards , /genetics
3.
PLoS One ; 16(2): e0246867, 2021.
Article in English | MEDLINE | ID: covidwho-1076273

ABSTRACT

Widespread diagnostic testing is needed to reduce transmission of COVID-19 and manage the pandemic. Effective mass screening requires robust and sensitive tests that reliably detect the SARS-CoV-2 virus, including asymptomatic and pre-symptomatic infections with a low viral count. Currently, the most accurate tests are based on detection of viral RNA by RT-PCR. We developed a method to process COVID-19 specimens that simplifies and increases the sensitivity of viral RNA detection by direct RT-qPCR, performed without RNA purification. In the method, termed Alkaline-Glycol Processing (AG Processing), a SARS-CoV-2-containing biological specimen, such as saliva or a swab-collected suspension, is processed at pH 12.2 to 12.8 for 5 min at room temperature. An aliquot of the AG-processed specimen is used for detection of SARS-CoV-2 RNA by direct RT-qPCR. AG processing effectively lyses viruses and reduces the effect of inhibitors of RT-PCR that are present in biological specimens. The sensitivity of detecting viral RNA using AG processing is on par with methods that include a viral RNA purification step. One copy of SARS-CoV-2 virus per reaction, equivalent to 300 copies per ml of saliva, is detectable in the AG-processed saliva. The LOD is 300 viral copies per ml of initial saliva specimen. AG processing works with saliva specimens or swab specimens collected into Universal Transport Medium, is compatible with heat treatment of saliva, and was confirmed to work with a range of CDC-approved RT-qPCR products and kits. Detection of SARS-CoV-2 RNA using AG processing with direct RT-qPCR provides a reliable and scalable diagnostic test for COVID-19 that can be integrated into a range of workflows, including automated settings.


Subject(s)
/diagnosis , Molecular Diagnostic Techniques/methods , RNA, Viral/genetics , /isolation & purification , Humans , Limit of Detection , Mass Screening , Reagent Kits, Diagnostic , Real-Time Polymerase Chain Reaction , Saliva/virology , Specimen Handling , Time Factors
4.
Genome Med ; 13(1): 21, 2021 02 09.
Article in English | MEDLINE | ID: covidwho-1076156

ABSTRACT

We present CoronaHiT, a platform and throughput flexible method for sequencing SARS-CoV-2 genomes (≤ 96 on MinION or > 96 on Illumina NextSeq) depending on changing requirements experienced during the pandemic. CoronaHiT uses transposase-based library preparation of ARTIC PCR products. Method performance was demonstrated by sequencing 2 plates containing 95 and 59 SARS-CoV-2 genomes on nanopore and Illumina platforms and comparing to the ARTIC LoCost nanopore method. Of the 154 samples sequenced using all 3 methods, ≥ 90% genome coverage was obtained for 64.3% using ARTIC LoCost, 71.4% using CoronaHiT-ONT and 76.6% using CoronaHiT-Illumina, with almost identical clustering on a maximum likelihood tree. This protocol will aid the rapid expansion of SARS-CoV-2 genome sequencing globally.


Subject(s)
/genetics , Genome, Viral/genetics , Pandemics , /genetics , /virology , High-Throughput Nucleotide Sequencing , Humans , RNA, Viral/genetics , Whole Genome Sequencing
5.
Curr Oncol ; 28(1): 847-852, 2021 02 08.
Article in English | MEDLINE | ID: covidwho-1069796

ABSTRACT

The pandemic of SARS-CoV-2 is a serious global challenge affecting millions of people worldwide. Cancer patients are at risk for infection exposure and serious complications. A prompt diagnosis of SARS-CoV-2 infection is crucial for the timely adoption of isolation measures and the appropriate management of cancer treatments. In lung cancer patients the symptoms of infection 19 may resemble those exhibited by the underlying oncologic condition, possibly leading to diagnostic overlap and delays. Moreover, cancer patients might display a prolonged positivity of nasopharyngeal RT-PCR assays for SARS-CoV-2, causing long interruptions or delay of cancer treatments. However, the association between the positivity of RT-PCR assays and the patient's infectivity remains uncertain. We describe the case of a patient with non-small cell lung cancer, and a severe ab extrinseco compression of the trachea, whose palliative radiotherapy was delayed because of the prolonged positivity of nasopharyngeal swabs for SARS-CoV-2. The patient did not show clinical symptoms suggestive of active infection, but the persistent positivity of RT-PCR assays imposed the continuation of isolation measures and the delay of radiotherapy for over two months. Finally, the negative result of SARS-CoV-2 viral culture allowed us to verify the absence of viral activity and to rule out the infectivity of the patient, who could finally continue her cancer treatment.


Subject(s)
/diagnosis , Carcinoma, Non-Small-Cell Lung/virology , Lung Neoplasms/virology , /isolation & purification , Aged , Carrier State/diagnosis , Female , Humans , Nasopharynx/virology , RNA, Viral/genetics , Time-to-Treatment
11.
Postepy Biochem ; 66(4): 323-335, 2020 12 31.
Article in Polish | MEDLINE | ID: covidwho-1064254

ABSTRACT

Coronaviruses are the causative agents of mild to severe respiratory and intestinal infections in humans. They are the largest RNA viruses, which genomes and encoded RNAs are known to fold into the highly-order structures that play essential roles in the viral replication and infectivity cycle. The recent outbreaks of new pathogenic coronaviruses steered researchers' attention into the possibility of targeting their RNAs directly with novel RNA-specific drugs and therapeutic strategies. In this manuscript, we highlight the recent biochemical and biophysical methodological advancements that yielded more in-depth insight into the structural and functional composition of coronaviruses cis-acting RNA motifs. We discuss the complexity of these RNA regulatory elements, their intermolecular interactions, post-transcriptional regulation, and their potential as druggable targets. We also indicate the location and function of unstructured and highly-conserved regions in coronaviruses RNA genomes representing viable aims for antisense oligonucleotide or CRISPR-based antiviral strategies.


Subject(s)
Coronavirus , Antiviral Agents , Coronavirus/genetics , Humans , Nucleotide Motifs , RNA, Viral/genetics , Virus Replication
14.
Viruses ; 13(1)2021 Jan 15.
Article in English | MEDLINE | ID: covidwho-1067778

ABSTRACT

Ferrets were experimentally inoculated with SARS-CoV-2 (severe acute respiratory syndrome (SARS)-related coronavirus 2) to assess infection dynamics and host response. During the resulting subclinical infection, viral RNA was monitored between 2 and 21 days post-inoculation (dpi), and reached a peak in the upper respiratory cavity between 4 and 6 dpi. Viral genomic sequence analysis in samples from three animals identified the Y453F nucleotide substitution relative to the inoculum. Viral RNA was also detected in environmental samples, specifically in swabs of ferret fur. Microscopy analysis revealed viral protein and RNA in upper respiratory tract tissues, notably in cells of the respiratory and olfactory mucosae of the nasal turbinates, including olfactory neuronal cells. Antibody responses to the spike and nucleoprotein were detected from 21 dpi, but virus-neutralizing activity was low. A second intranasal inoculation (re-exposure) of two ferrets after a 17-day interval did not produce re-initiation of viral RNA shedding, but did amplify the humoral response in one animal. Therefore, ferrets can be experimentally infected with SARS-CoV-2 to model human asymptomatic infection.


Subject(s)
Asymptomatic Diseases , Disease Models, Animal , /physiology , Animals , Antibodies, Viral/blood , Antibodies, Viral/immunology , /pathology , Female , Ferrets , Genome, Viral/genetics , Mutation , Nasal Mucosa/virology , RNA, Viral/genetics , Viral Load , Virus Shedding
15.
Int J Mol Sci ; 22(3)2021 Jan 20.
Article in English | MEDLINE | ID: covidwho-1067753

ABSTRACT

The COVID-19 pandemic caused by the SARS-CoV-2 virus, which first emerged in December 2019, represents an ongoing global public health emergency. Here, we developed an improved and highly sensitive approach to SARS-CoV-2 detection via coupling bioluminescence in real-time (BART) and reverse-transcriptase loop-mediated amplification (RT-LAMP) protocols (RT-LAMP-BART) and was also compatible with a digital LAMP system (Rainsuit), which did not allow for real-time quantification but did, nonetheless, facilitate absolute quantification with a comparable detection limit of 104 copies/mL. Through improving RNA availability in samples to ensure the target RNA present in reaction, we additionally developed a simulated digital RT-LAMP approach using this same principle to enlarge the overall reaction volume and to achieve real-time detection with a limit of detection of 10 copies/mL, and with further improvements in the overall dynamic range of this assay system being achieved through additional optimization.


Subject(s)
/virology , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , RNA, Viral/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Viral Proteins/genetics , Humans , Limit of Detection , Luminescent Measurements/methods , Polyproteins/genetics , RNA, Viral/analysis , Real-Time Polymerase Chain Reaction/methods , Reverse Transcription
16.
Genes (Basel) ; 12(1)2021 Jan 13.
Article in English | MEDLINE | ID: covidwho-1067701

ABSTRACT

In February 2020, our laboratory started to offer a RT-qPCR assay for the qualitative detection of severe acute respiratory syndrome coronavirus 2. A few months after the assay was released to our patients, some materials, reagents, and equipment became in short supply. Alternative protocols were necessary in order to avoid stopping testing to the population. However, the suitability of these alternatives needs to be validated before their use. Here, we investigated if saliva is a reliable alternative specimen to nasopharyngeal swabs; if 0.45% saline is a reliable alternative to guanidine hydrochloride as a collection viral transport media; the stability of SARS-COV-2 in guanidine hydrochloride and in 0.45% saline for 10 and 50 days at room temperature; and if the primers/probe concentration and thermocycling times could be reduced so as to overcome the short supply of these reagents and equipment, without a significant loss of the assay performance. We found that saliva is not an appropriated specimen for our method-nasopharyngeal swabs perform better. Saline (0.45%) and guanidine hydrochloride have a similar SARS-CoV-2 diagnostic capability as tube additives. Reliable SARS-CoV-2 RNA detection can be performed after sample storage for 10 days at room temperature (18-23 °C) in both 0.45% saline and guanidine hydrochloride. Using synthetic RNA, and decreasing the concentration of primers by five-fold and probes by 2.5-fold, changed the assay limit of detection (LOD) from 7.2 copies/reaction to 23.7 copies/reaction and the subsequent reducing of thermocycling times changed the assay LOD from 23.7 copies/reaction to 44.2 copies/reaction. However, using real clinical samples with Cq values ranging from ~12.15 to ~36.46, the results of the three tested conditions were almost identical. These alterations will not affect the vast majority of diagnostics and increase the daily testing capability in 30% and increase primers and probe stocks in 500% and 250%, respectively. Taken together, the alternative protocols described here overcome the short supply of tubes, reagents and equipment during the SARS-CoV-2 pandemic, avoiding the collapse of test offering for the population: 105,757 samples were processed, and 25,156 SARS-CoV-2 diagnostics were performed from 9 May 2020 to 30 June 2020.


Subject(s)
RNA, Viral , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , /diagnosis , Female , Humans , Male , RNA, Viral/genetics , RNA, Viral/metabolism , /metabolism
17.
Euro Surveill ; 26(5)2021 02.
Article in English | MEDLINE | ID: covidwho-1067624

ABSTRACT

In June-November 2020, SARS-CoV-2-infected mink were detected in 290 of 1,147 Danish mink farms. In North Denmark Region, 30% (324/1,092) of people found connected to mink farms tested SARS-CoV-2-PCR-positive and approximately 27% (95% confidence interval (CI): 25-30) of SARS-CoV-2-strains from humans in the community were mink-associated. Measures proved insufficient to mitigate spread. On 4 November, the government ordered culling of all Danish mink. Farmed mink constitute a potential virus reservoir challenging pandemic control.


Subject(s)
Animals, Wild/virology , /veterinary , Disease Outbreaks/veterinary , Disease Reservoirs/veterinary , Disease Transmission, Infectious/veterinary , Mink/virology , Pandemics/veterinary , /isolation & purification , /transmission , Animals , /virology , Denmark/epidemiology , Disease Outbreaks/statistics & numerical data , Disease Reservoirs/virology , Farms , Genes, Viral , Humans , Incidence , Polymerase Chain Reaction , Public Health , RNA, Viral/analysis , RNA, Viral/genetics , /virology , Whole Genome Sequencing , Zoonoses/transmission , Zoonoses/virology
18.
PLoS One ; 15(12): e0243959, 2020.
Article in English | MEDLINE | ID: covidwho-1067398

ABSTRACT

There has been significant concern regarding fertility and reproductive outcomes during the SARS-CoV2 pandemic. Recent data suggests a high concentration of SARS-Cov2 receptors, ACE2 or TMPRSS2, in nasal epithelium and cornea, which explains person-to-person transmission. We investigated the prevalence of SARS-CoV2 receptors among reproductive tissues by exploring the single-cell sequencing datasets from uterus, myometrium, ovary, fallopian tube, and breast epithelium. We did not detect significant expression of either ACE2 or TMPRSS2 in the normal human myometrium, uterus, ovaries, fallopian tube, or breast. Furthermore, none of the cell types in the female reproductive organs we investigated, showed the co-expression of ACE2 with proteases, TMPRSS2, Cathepsin B (CTSB), and Cathepsin L (CTSL) known to facilitate the entry of SARS2-CoV2 into the host cell. These results suggest that myometrium, uterus, ovaries, fallopian tube, and breast are unlikely to be susceptible to infection by SARS-CoV2.


Subject(s)
/genetics , Cathepsin B/genetics , Cathepsin L/genetics , Serine Endopeptidases/genetics , /metabolism , Breast/metabolism , Breast/virology , /transmission , Epithelium/metabolism , Epithelium/virology , Fallopian Tubes/metabolism , Fallopian Tubes/virology , Female , Fertility/genetics , High-Throughput Nucleotide Sequencing , Humans , Myometrium/metabolism , Myometrium/virology , Ovary/metabolism , Ovary/virology , RNA, Viral/genetics , RNA, Viral/isolation & purification , Reproductive Tract Infections/genetics , Reproductive Tract Infections/virology , Serine Endopeptidases/metabolism , Single-Cell Analysis , Uterus/metabolism , Uterus/virology
19.
Ann Clin Microbiol Antimicrob ; 20(1): 8, 2021 Jan 18.
Article in English | MEDLINE | ID: covidwho-1067240

ABSTRACT

The Severe Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has gained research attention worldwide, given the current pandemic. Nevertheless, a previous zoonotic and highly pathogenic coronavirus, the Middle East Respiratory Syndrome coronavirus (MERS-CoV), is still causing concern, especially in Saudi Arabia and neighbour countries. The MERS-CoV has been reported from respiratory samples in more than 27 countries, and around 2500 cases have been reported with an approximate fatality rate of 35%. After its emergence in 2012 intermittent, sporadic cases, nosocomial infections and many community clusters of MERS continued to occur in many countries. Human-to-human transmission resulted in the large outbreaks in Saudi Arabia. The inherent genetic variability among various clads of the MERS-CoV might have probably paved the events of cross-species transmission along with changes in the inter-species and intra-species tropism. The current review is drafted using an extensive review of literature on various databases, selecting of publications irrespective of favouring or opposing, assessing the merit of study, the abstraction of data and analysing data. The genome of MERS-CoV contains around thirty thousand nucleotides having seven predicted open reading frames. Spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins are the four main structural proteins. The surface located spike protein (S) of betacoronaviruses has been established to be one of the significant factors in their zoonotic transmission through virus-receptor recognition mediation and subsequent initiation of viral infection. Three regions in Saudi Arabia (KSA), Eastern Province, Riyadh and Makkah were affected severely. The epidemic progression had been the highest in 2014 in Makkah and Riyadh and Eastern Province in 2013. With a lurking epidemic scare, there is a crucial need for effective therapeutic and immunological remedies constructed on sound molecular investigations.


Subject(s)
Antiviral Agents/therapeutic use , Coronavirus Infections/drug therapy , /genetics , Middle East Respiratory Syndrome Coronavirus/genetics , Spike Glycoprotein, Coronavirus/genetics , /genetics , Coronavirus Infections/epidemiology , Coronavirus Infections/transmission , Cross Infection/epidemiology , Cross Infection/virology , Disease Outbreaks , Humans , Middle East Respiratory Syndrome Coronavirus/isolation & purification , Phylogeny , RNA, Viral/genetics , Saudi Arabia/epidemiology
20.
Proc Natl Acad Sci U S A ; 118(8)2021 02 23.
Article in English | MEDLINE | ID: covidwho-1066042

ABSTRACT

The characterization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral kinetics in hospitalized patients and its association with mortality is unknown. We analyzed death and nasopharyngeal viral kinetics in 655 hospitalized patients from the prospective French COVID cohort. The model predicted a median peak viral load that coincided with symptom onset. Patients with age ≥65 y had a smaller loss rate of infected cells, leading to a delayed median time to viral clearance occurring 16 d after symptom onset as compared to 13 d in younger patients (P < 10-4). In multivariate analysis, the risk factors associated with mortality were age ≥65 y, male gender, and presence of chronic pulmonary disease (hazard ratio [HR] > 2.0). Using a joint model, viral dynamics after hospital admission was an independent predictor of mortality (HR = 1.31, P < 10-3). Finally, we used our model to simulate the effects of effective pharmacological interventions on time to viral clearance and mortality. A treatment able to reduce viral production by 90% upon hospital admission would shorten the time to viral clearance by 2.0 and 2.9 d in patients of age <65 y and ≥65 y, respectively. Assuming that the association between viral dynamics and mortality would remain similar to that observed in our population, this could translate into a reduction of mortality from 19 to 14% in patients of age ≥65 y with risk factors. Our results show that viral dynamics is associated with mortality in hospitalized patients. Strategies aiming to reduce viral load could have an effect on mortality rate in this population.


Subject(s)
/mortality , Models, Theoretical , Nasopharynx/virology , RNA, Viral/analysis , Viral Load , Aged , Antibodies, Viral/blood , /epidemiology , Female , France/epidemiology , Hospitalization , Humans , Kinetics , Male , Prognosis , Prospective Studies , RNA, Viral/genetics , Risk Factors , Survival Rate
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