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1.
PLoS One ; 16(4): e0250319, 2021.
Article in English | MEDLINE | ID: covidwho-1833525

ABSTRACT

Projections of the stage of the Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) pandemic and local, regional and national public health policies to limit coronavirus spread as well as "reopen" cities and states, are best informed by serum neutralizing antibody titers measured by reproducible, high throughput, and statically credible antibody (Ab) assays. To date, a myriad of Ab tests, both available and FDA authorized for emergency, has led to confusion rather than insight per se. The present study reports the results of a rapid, point-in-time 1,000-person cohort study using serial blood donors in the New York City metropolitan area (NYC) using multiple serological tests, including enzyme-linked immunosorbent assays (ELISAs) and high throughput serological assays (HTSAs). These were then tested and associated with assays for neutralizing Ab (NAb). Of the 1,000 NYC blood donor samples in late June and early July 2020, 12.1% and 10.9% were seropositive using the Ortho Total Ig and the Abbott IgG HTSA assays, respectively. These serological assays correlated with neutralization activity specific to SARS-CoV-2. The data reported herein suggest that seroconversion in this population occurred in approximately 1 in 8 blood donors from the beginning of the pandemic in NYC (considered March 1, 2020). These findings deviate with an earlier seroprevalence study in NYC showing 13.7% positivity. Collectively however, these data demonstrate that a low number of individuals have serologic evidence of infection during this "first wave" and suggest that the notion of "herd immunity" at rates of ~60% or higher are not near. Furthermore, the data presented herein show that the nature of the Ab-based immunity is not invariably associated with the development of NAb. While the blood donor population may not mimic precisely the NYC population as a whole, rapid assessment of seroprevalence in this cohort and serial reassessment could aid public health decision making.


Subject(s)
COVID-19/epidemiology , SARS-CoV-2/immunology , Adolescent , Adult , Aged , Aged, 80 and over , Antibodies, Neutralizing/blood , Antibodies, Viral/immunology , Blood Donors , COVID-19/immunology , Cohort Studies , Enzyme-Linked Immunosorbent Assay/methods , Female , Humans , Immunoglobulin G/blood , Male , Middle Aged , New York City/epidemiology , SARS-CoV-2/pathogenicity , Sensitivity and Specificity , Seroconversion/physiology , Seroepidemiologic Studies , Serologic Tests/methods , Spike Glycoprotein, Coronavirus/immunology
2.
Clin Infect Dis ; 74(4): 584-590, 2022 03 01.
Article in English | MEDLINE | ID: covidwho-1709326

ABSTRACT

BACKGROUND: With limited severe acute respiratory syndrome coronavirus (SARS-CoV-2) testing capacity in the United States at the start of the epidemic (January-March 2020), testing was focused on symptomatic patients with a travel history throughout February, obscuring the picture of SARS-CoV-2 seeding and community transmission. We sought to identify individuals with SARS-CoV-2 antibodies in the early weeks of the US epidemic. METHODS: All of Us study participants in all 50 US states provided blood specimens during study visits from 2 January to 18 March 2020. Participants were considered seropositive if they tested positive for SARS-CoV-2 immunoglobulin G (IgG) antibodies with the Abbott Architect SARS-CoV-2 IgG enzyme-linked immunosorbent assay (ELISA) and the EUROIMMUN SARS-CoV-2 ELISA in a sequential testing algorithm. The sensitivity and specificity of these ELISAs and the net sensitivity and specificity of the sequential testing algorithm were estimated, along with 95% confidence intervals (CIs). RESULTS: The estimated sensitivities of the Abbott and EUROIMMUN assays were 100% (107 of 107 [95% CI: 96.6%-100%]) and 90.7% (97 of 107 [83.5%-95.4%]), respectively, and the estimated specificities were 99.5% (995 of 1000 [98.8%-99.8%]) and 99.7% (997 of 1000 [99.1%-99.9%]), respectively. The net sensitivity and specificity of our sequential testing algorithm were 90.7% (97 of 107 [95% CI: 83.5%-95.4%]) and 100.0% (1000 of 1000 [99.6%-100%]), respectively. Of the 24 079 study participants with blood specimens from 2 January to 18 March 2020, 9 were seropositive, 7 before the first confirmed case in the states of Illinois, Massachusetts, Wisconsin, Pennsylvania, and Mississippi. CONCLUSIONS: Our findings identified SARS-CoV-2 infections weeks before the first recognized cases in 5 US states.


Subject(s)
COVID-19 , Population Health , Antibodies, Viral , COVID-19/diagnosis , Enzyme-Linked Immunosorbent Assay , Humans , Immunoglobulin G , SARS-CoV-2 , Sensitivity and Specificity
3.
J Ayurveda Integr Med ; 13(2): 100449, 2022.
Article in English | MEDLINE | ID: covidwho-1593865

ABSTRACT

BACKGROUND: The recent outbreak of the novel SARS-CoV-2 across the globe and the absence of specific drug against this virus lead the scientific community to look into some alternative indigenous treatments. India as a hub of Ayurvedic and medicinal plants can shed light on its treatment using specific active bio-molecules from these plants. OBJECTIVES: Keeping our herbal resources in mind, we were interested to inquire whether some phytochemicals from Indian spices and medicinal plants can be used as alternative therapeutic agents in contrast to synthetic drugs. MATERIALS AND METHODS: We used in silico molecular docking approach to test whether bioactive molecules of herbal origin such as hyperoside, nimbaflavone, ursolic acid, 6-gingerol, 6-shogaol and 6-paradol, curcumin, catechins and epigallocatechin, α-Hederin, piperine could bind and potentially block the Mproenzyme of the SARS-CoV-2 virus. RESULTS: Ursolic acid showed the highest docking score (-8.7 kcal/mol) followed by hyperoside (-8.6 kcal/mol), α-Hederin (-8.5 kcal/mol) and nimbaflavone (-8.0 kcal/mol). epigallocatechin, catechins, and curcumin also exhibited high binding affinity (Docking score -7.3, -7.1 and -7.1 kcal/mol) with the Mpro. The remaining tested phytochemicals exhibited moderate binding and inhibitory effects. CONCLUSION: This finding provides a basis for biochemical assay of tested bioactive molecules on SARS-CoV-2 virus.

4.
Clin Chem Lab Med ; 59(8): 1463-1467, 2021 07 27.
Article in English | MEDLINE | ID: covidwho-1546996

ABSTRACT

OBJECTIVES: COVID-19 has brought about tests from many manufacturers. While molecular and rapid antigen tests are targeted for early diagnosis, immunoassays have a larger role in epidemiological studies, understanding longitudinal immunity, and in vaccine development and response. METHODS: The performance of the LIAISON® SARS-CoV-2 TrimericS IgG assay was evaluated against the Beckman ACCESS SARS-CoV-2 IgG assay in New Mexico, and against the Siemens ADVIA Centaur COV2G assay in New York. Discordant samples were parsed using a microneutralization assay. RESULTS: A SARS-CoV-2 antibody positivity rate of 23.8% was observed in the samples tested in New York (September 2020), while in the same month the positivity rate was 1.5% in New Mexico. Positive and negative agreement were 67.6% (95% CI 49.5-82.6%) and 99.8% (95% CI 99.5-99.9%), respectively, with the Beckman test, and 98.0% (95% CI 95.7-99.3%) and 94.8% (95% CI 93.4-96.0%), respectively, with the Siemens test. Receiver operating characteristic analysis for the detection of SARS-CoV-2 antibodies discloses an AUC, area under the curve, of 0.996 (95% CI 0.992-0.999) for the LIAISON® SARS-CoV-2 TrimericS IgG assay. The criterion associated to the Youden Index was determined to be >12.9 kAU/L with a sensitivity of 99.44% and a specificity of 99.82%. CONCLUSIONS: The LIAISON® SARS-CoV-2 TrimericS IgG assay is highly sensitive and specific. The balance of these parameters, without emphasis on high specificity alone, is particularly important when applied to high prevalence populations, where a highly sensitive assay will result in reporting a lower number of false negative subjects.


Subject(s)
Antibodies, Viral/blood , COVID-19/diagnosis , Immunoassay/methods , Immunoglobulin G/blood , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/immunology , Area Under Curve , Automation , COVID-19/virology , Humans , ROC Curve , Reagent Kits, Diagnostic , SARS-CoV-2/isolation & purification , Sensitivity and Specificity
5.
Wellcome Open Res ; 6: 9, 2021.
Article in English | MEDLINE | ID: covidwho-1502788

ABSTRACT

The ongoing pandemic of SARS-CoV-2 calls for rapid and cost-effective methods to accurately identify infected individuals. The vast majority of patient samples is assessed for viral RNA presence by RT-qPCR. Our biomedical research institute, in collaboration between partner hospitals and an accredited clinical diagnostic laboratory, established a diagnostic testing pipeline that has reported on more than 252,000 RT-qPCR results since its commencement at the beginning of April 2020. However, due to ongoing demand and competition for critical resources, alternative testing strategies were sought. In this work, we present a clinically-validated procedure for high-throughput SARS-CoV-2 detection by RT-LAMP in 25 minutes that is robust, reliable, repeatable, sensitive, specific, and inexpensive.

6.
Clin Infect Dis ; 73(9): e2853-e2860, 2021 11 02.
Article in English | MEDLINE | ID: covidwho-1501011

ABSTRACT

BACKGROUND: The objective of this study was to perform a seroprevalence survey on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among Danish healthcare workers to identify high-risk groups. METHODS: All healthcare workers and administrative personnel at the 7 hospitals, prehospital services, and specialist practitioner clinics in the Central Denmark Region were invited to be tested by a commercial SARS-CoV-2 total antibody enzyme-linked immunosorbent assay (Wantai Biological Pharmacy Enterprise Co, Ltd, Beijing, China). RESULTS: A total of 25 950 participants were invited. Of these, 17 971 had samples available for SARS-CoV-2 antibody testing. After adjustment for assay sensitivity and specificity, the overall seroprevalence was 3.4% (95% confidence interval [CI], 2.5%-3.8%). The seroprevalence was higher in the western part of the region than in the eastern part (11.9% vs 1.2%; difference: 10.7 percentage points [95% CI, 9.5-12.2]). In the high-prevalence area, the emergency departments had the highest seroprevalence (29.7%), whereas departments without patients or with limited patient contact had the lowest seroprevalence (2.2%). Among the total 668 seropositive participants, 433 (64.8%) had previously been tested for SARS-CoV-2 RNA, and 50.0% had a positive reverse-transcription polymerase chain reaction (PCR) result. CONCLUSIONS: We found large differences in the prevalence of SARS-CoV-2 antibodies in staff working in the healthcare sector within a small geographical area of Denmark. Half of all seropositive staff had been tested positive by PCR prior to this survey. This study raises awareness of precautions that should be taken to avoid in-hospital transmission. Regular testing of healthcare workers for SARS-CoV-2 should be considered to identify areas with increased transmission.


Subject(s)
COVID-19 , Emergency Medical Services , Administrative Personnel , Antibodies, Viral , Delivery of Health Care , Denmark/epidemiology , Health Personnel , Hospitals , Humans , RNA, Viral , SARS-CoV-2 , Seroepidemiologic Studies
7.
J Med Virol ; 93(10): 5816-5824, 2021 10.
Article in English | MEDLINE | ID: covidwho-1453607

ABSTRACT

Serological testing for anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies is used to detect ongoing or past SARS-CoV-2 infections. To study the kinetics of anti-SARS-CoV-2 antibodies and to assess the diagnostic performances of eight serological assays, we used 129 serum samples collected on known days post symptom onset (dpso) from 42 patients with polymerase chain reaction-confirmed coronavirus disease 2019 (COVID-19) and 54 serum samples from healthy blood donors, and children infected with seasonal coronaviruses. The sera were analyzed for the presence of immunoglobulin G (IgG), immunoglobulin M (IgM), and immunoglobulin A (IgA) antibodies using indirect immunofluorescence testing (IIFT) based on SARS-CoV-2-infected cells. They were further tested for antibodies against the S1 domain of the SARS-CoV-2 spike protein (IgG, IgA) and against the viral nucleocapsid protein (IgG, IgM) using enzyme-linked immunosorbent assays. The assay specificities were 94.4%-100%. The sensitivities varied largely between assays, reflecting their respective purposes. The sensitivities of IgA and IgM assays were the highest between 11 and 20 dpso, whereas the sensitivities of IgG assays peaked between 20 and 60 dpso. IIFT showed the highest sensitivities due to the use of the whole SARS-CoV-2 as substrate and provided information on whether or not the individual has been infected with SARS-CoV-2. Enzyme-linked immunosorbent assays provided further information about both the prevalence and concentration of specific antibodies against selected antigens of SARS-CoV-2.


Subject(s)
Antibodies, Viral/blood , COVID-19 Serological Testing , COVID-19/blood , SARS-CoV-2/immunology , Adult , Aged , Aged, 80 and over , Antibodies, Viral/immunology , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/immunology , Enzyme-Linked Immunosorbent Assay , Female , Fluorescent Antibody Technique, Indirect , Humans , Immunoglobulin Isotypes/blood , Kinetics , Male , Middle Aged , Phosphoproteins/immunology , SARS-CoV-2/isolation & purification , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/immunology
8.
Jpn J Infect Dis ; 74(5): 465-472, 2021 Sep 22.
Article in English | MEDLINE | ID: covidwho-1436361

ABSTRACT

Soon after the 2019 outbreak of coronavirus disease 2019 in Wuhan, China, a protocol for real-time RT-PCR assay detection of severe acute respiratory syndrome coronavirus (SARS-CoV-2) was established by the National Institute of Infectious Diseases (NIID) in Japan. The protocol used Charité's nucleocapsid (Sarbeco-N) and NIID nucleocapsid (NIID-N2) assays. During the following months, SARS-CoV-2 spread and caused a global pandemic, and various SARS-CoV-2 sequences were registered in public databases, such as the Global Initiative on Sharing All Influenza Data (GISAID). In this study, we evaluated the S2 assay (NIID-S2) that was newly developed to replace the Sarbeco-N assay and the performance of the NIID-N2 and NIID-S2 assays, referring to mismatches in the primer/probe targeted region. We found that the analytical sensitivity and specificity of the NIID-S2 set were comparable to those of the NIID-N2 assay, and the detection rate for clinical specimens was identical to that of the NIID-N2 assay. Furthermore, among the available sequences (approximately 192,000), the NIID-N2 and NIID-S2 sets had 2.6% and 1.2% mismatched sequences, respectively, although most of these mismatches did not affect the amplification efficiency, except the 3' end of the NIID-N2 forward primer. These findings indicate that the previously developed NIID-N2 assay is suitable for the detection of SARS-CoV-2 with support from the newly developed NIID-S2 set.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , SARS-CoV-2/isolation & purification , Coronavirus Nucleocapsid Proteins/genetics , DNA Primers/genetics , Humans , Japan , Phosphoproteins/genetics , RNA, Viral/analysis , RNA, Viral/genetics , SARS-CoV-2/genetics , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/genetics
9.
Ghana Med J ; 54(4 Suppl): 77-85, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-1436198

ABSTRACT

BACKGROUND: A novel coronavirus, SARS-CoV-2 is currently causing a worldwide pandemic. The first cases of SARS-CoV-2 infection were recorded in Ghana on March 12, 2020. Since then, the country has been combatting countrywide community spread. This report describes how the Virology Department, Noguchi Memorial Institute for Medical Research (NMIMR) is supporting the Ghana Health Service (GHS) to diagnose infections with this virus in Ghana. METHODS: The National Influenza Centre (NIC) in the Virology Department of the NMIMR, adopted real-time Polymerase Chain Reaction (rRT-PCR) assays for the diagnosis of the SARS-CoV-2 in January 2020. Samples from suspected cases and contact tracing across Ghana were received and processed for SARS-CoV-2. Samples were 'pooled' to enable simultaneous batch testing of samples without reduced sensitivity. OUTCOMES: From February 3 to August 21, the NMIMR processed 283 946 (10%) samples. Highest number of cases were reported in June when the GHS embarked on targeted contact tracing which led to an increase in number of samples processed daily, peaking at over 7,000 samples daily. There were several issues to overcome including rapid consumption of reagents and consumables. Testing however continued successfully due to revised procedures, additional equipment and improved pipeline of laboratory supplies. Test results are now provided within 24 to 48 hours of sample submission enabling more effective response and containment. CONCLUSION: Following the identification of the first cases of SARS-CoV-2infection by the NMIMR, the Institute has trained other centres and supported the ramping up of molecular testing capacity in Ghana. This provides a blueprint to enable Ghana to mitigate further epidemics and pandemics. FUNDING: The laboratory work was supported with materials from the Ghana Health Service Ministry of Health, the US Naval Medical Research Unit #3, the World Health Organization, the Jack Ma Foundation and the University of Ghana Noguchi Memorial Institute for Medical Research. Other research projects hosted by the Noguchi Memorial Institute for Medical Research contributed reagents and laboratory consumables. The funders had no role in the preparation of this manuscript.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Infection Control/methods , Population Surveillance , SARS-CoV-2/isolation & purification , COVID-19/epidemiology , Contact Tracing/methods , Contact Tracing/statistics & numerical data , Ghana/epidemiology , Humans , National Health Programs , SARS-CoV-2/genetics
10.
Microorganisms ; 9(3)2021 Mar 08.
Article in English | MEDLINE | ID: covidwho-1389449

ABSTRACT

Several studies have described the long-term kinetics of anti-SARS-CoV-2 antibodies but long-term follow-up data, i.e., >6 months, are still sparse. Additionally, the literature is inconsistent regarding the waning effect of the serological response. The aim of this study was to explore the temporal dynamic changes of the immune response after SARS-CoV-2 infection in hospitalized and non-hospitalized symptomatic patients over a period of 10 months. Six different analytical kits for SARS-CoV-2 antibody detection were used. Positivity rates, inter-assay agreement and kinetic models were determined. A high inter-individual and an inter-methodology variability was observed. Assays targeting total antibodies presented higher positivity rates and reached the highest positivity rates sooner compared with assays directed against IgG. The inter-assay agreement was also higher between these assays. The stratification by disease severity showed a much-elevated serological response in hospitalized versus non-hospitalized patients in all assays. In this 10-month follow-up study, serological assays showed a clinically significant difference to detect past SARS-CoV-2 infection with total antibody assays presenting the highest positivity rates. The waning effect reported in several studies should be interpreted with caution because it could depend on the assay considered.

11.
Transfusion ; 61(1): 17-23, 2021 01.
Article in English | MEDLINE | ID: covidwho-1388418

ABSTRACT

BACKGROUND: The transfer of passive immunity with convalescent plasma is a promising strategy for treatment and prevention of COVID-19, but donors with a history of nonsevere disease are serologically heterogenous. The relationship between SARS-Cov-2 antigen-binding activity and neutralization activity in this population of donors has not been defined. STUDY DESIGN AND METHODS: Convalescent plasma units from 47 individuals with a history of nonsevere COVID-19 were assessed for antigen-binding activity of using three clinical diagnostic serology assays (Beckman, DiaSorin, and Roche) with different SARS-CoV-2 targets. These results were compared with functional neutralization activity using a fluorescent reporter strain of SARS-CoV-2 in a microwell assay. RESULTS: Positive correlations of varying strength (Spearman r = 0.37-0.52) between antigen binding and viral neutralization were identified. Donors age 48 to 75 years had the highest neutralization activity. Units in the highest tertile of binding activity for each assay were enriched (75%-82%) for those with the highest levels of neutralization. CONCLUSION: The strength of the relationship between antigen-binding activity and neutralization varies depending on the clinical assay used. Units in the highest tertile of binding activity for each assay are predominantly comprised of those with the greatest neutralization activity.


Subject(s)
SARS-CoV-2/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , COVID-19/therapy , COVID-19 Serological Testing , Enzyme-Linked Immunosorbent Assay , Humans , Immunization, Passive , Immunoglobulin G/immunology , SARS-CoV-2/pathogenicity , Serologic Tests
12.
J AOAC Int ; 104(4): 872-888, 2021 Aug 20.
Article in English | MEDLINE | ID: covidwho-1387921

ABSTRACT

BACKGROUND: The Eurofins GeneScan Technologies' VIRSeek SARS-CoV-2 Mplex kit is a RT (reverse transcription) real-time polymerase chain reaction (RT-qPCR) assay for the detection of two targets on the N-gene (nucleocapsid) of SARS-CoV-2. An extraction control, that allows monitoring of the extraction procedure and PCR inhibition, is included. OBJECTIVE: In silico analysis and wet testing showed inclusivity and exclusivity of the assay. The complete workflow starting from surface swabbing (VIRSeek PATHOSwab kit), RNA extraction (VIRSeek RNAExtractor), RT-PCR (VIRSeek SARS-CoV-2 Mplex), and evaluation with FastFinder was validated in comparison to the CDC method for detection of SARS-CoV-2 on stainless steel. METHOD: In silico analysis was performed by using the MFOLD online program. The matrix study was performed for stainless steel inoculated with SARS-CoV-2 isolated from the first documented US case of a traveler from Wuhan, China. RESULTS: For inclusivity, 15 764 sequences were analyzed and all mismatches (0.37% of the sequences had single mismatches) were considered non-critical. Cross reactivity for closely related viruses and background organisms was performed, resulting in correct exclusion of all. No significant differences were observed for the probability of detection (POD) study when comparing to the CDC method. CONCLUSIONS: Results of the inclusivity and exclusivity study show that the assay is specific for detection of SARS-CoV-2. The POD study showed no statistically significant difference compared to the CDC reference method, results were identical for the uninoculated and the high level. For the fractional recovery level, the candidate method detected 9/17 samples leading to a POD of 0.47, the reference method detected 11/20 samples leading to a POD of 0.55. HIGHLIGHT: The complete workflow starting from swabbing of the surface (VIRSeek PATHOSwab kit), RNA extraction (VIRSeek RNAExtractor), RT-PCR (VIRSeek SARS CoV-2 Mplex) and evaluation with FastFinder was validated in comparison to the US Centers for Disease Control and Prevention method for detection of SARS-CoV-2 on Stainless Steel.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction , Sensitivity and Specificity , Stainless Steel
13.
Viruses ; 12(5)2020 05 06.
Article in English | MEDLINE | ID: covidwho-1389513

ABSTRACT

SARS-CoV-2 enters cells using its Spike protein, which is also the main target of neutralizing antibodies. Therefore, assays to measure how antibodies and sera affect Spike-mediated viral infection are important for studying immunity. Because SARS-CoV-2 is a biosafety-level-3 virus, one way to simplify such assays is to pseudotype biosafety-level-2 viral particles with Spike. Such pseudotyping has now been described for single-cycle lentiviral, retroviral, and vesicular stomatitis virus (VSV) particles, but the reagents and protocols are not widely available. Here, we detailed how to effectively pseudotype lentiviral particles with SARS-CoV-2 Spike and infect 293T cells engineered to express the SARS-CoV-2 receptor, ACE2. We also made all the key experimental reagents available in the BEI Resources repository of ATCC and the NIH. Furthermore, we demonstrated how these pseudotyped lentiviral particles could be used to measure the neutralizing activity of human sera or plasma against SARS-CoV-2 in convenient luciferase-based assays, thereby providing a valuable complement to ELISA-based methods that measure antibody binding rather than neutralization.


Subject(s)
Antibodies, Viral/immunology , Neutralization Tests/methods , Spike Glycoprotein, Coronavirus/analysis , Angiotensin-Converting Enzyme 2 , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Containment of Biohazards , HEK293 Cells , Humans , Lentivirus , Peptidyl-Dipeptidase A/metabolism , Plasma/immunology
14.
Front Plant Sci ; 11: 601316, 2020.
Article in English | MEDLINE | ID: covidwho-1389236

ABSTRACT

We report to use the main protease (Mpro) of SARS-Cov-2 to screen plant flavan-3-ols and proanthocyanidins. Twelve compounds, (-)-afzelechin (AF), (-)-epiafzelechin (EAF), (+)-catechin (CA), (-)-epicatechin (EC), (+)-gallocatechin (GC), (-)-epigallocatechin (EGC), (+)-catechin-3-O-gallate (CAG), (-)-epicatechin-3-O-gallate (ECG), (-)-gallocatechin-3-O-gallate (GCG), (-)-epigallocatechin-3-O-gallate (EGCG), procyanidin A2 (PA2), and procyanidin B2 (PB2), were selected for docking simulation. The resulting data predicted that all 12 metabolites could bind to Mpro. The affinity scores of PA2 and PB2 were predicted to be -9.2, followed by ECG, GCG, EGCG, and CAG, -8.3 to -8.7, and then six flavan-3-ol aglycones, -7.0 to -7.7. Docking characterization predicted that these compounds bound to three or four subsites (S1, S1', S2, and S4) in the binding pocket of Mpro via different spatial ways and various formation of one to four hydrogen bonds. In vitro analysis with 10 available compounds showed that CAG, ECG, GCG, EGCG, and PB2 inhibited the Mpro activity with an IC50 value, 2.98 ± 0.21, 5.21 ± 0.5, 6.38 ± 0.5, 7.51 ± 0.21, and 75.3 ± 1.29 µM, respectively, while CA, EC, EGC, GC, and PA2 did not have inhibitory activities. To further substantiate the inhibitory activities, extracts prepared from green tea (GT), two muscadine grapes (MG), cacao, and dark chocolate (DC), which are rich in CAG, ECG, GAG, EGCG, or/and PB2, were used for inhibitory assay. The resulting data showed that GT, two MG, cacao, and DC extracts inhibited the Mpro activity with an IC50 value, 2.84 ± 0.25, 29.54 ± 0.41, 29.93 ± 0.83, 153.3 ± 47.3, and 256.39 ± 66.3 µg/ml, respectively. These findings indicate that on the one hand, the structural features of flavan-3-ols are closely associated with the affinity scores; on the other hand, the galloylation and oligomeric types of flavan-3-ols are critical in creating the inhibitory activity against the Mpro activity.

15.
Front Med (Lausanne) ; 7: 603996, 2020.
Article in English | MEDLINE | ID: covidwho-1389195

ABSTRACT

Seroprevalence studies are crucial both for estimating the prevalence of SARS-CoV-2 exposure and to provide a measure for the efficiency of the confinement measures. Portuguese universities were closed on March 16th 2020, when Portugal only registered 62 SARS-CoV-2 infection cases per million. We have validated a SARS-CoV-2 ELISA assay to a stabilized full-length spike protein using 216 pre-pandemic and 19 molecularly diagnosed SARS-CoV-2 positive individual's samples. At NOVA University of Lisbon, presential work was partially resumed on May 25th with staggered schedules. From June 15th to 30th, 3-4 weeks after the easing of confinement measures, we screened 1,636 collaborators of NOVA university of Lisbon for the presence of SARS-CoV-2 spike specific IgA and IgG antibodies. We found that spike-specific IgG in 50 of 1,636 participants (3.0%), none of which had anti-spike IgA antibodies. As participants self-reported as asymptomatic or paucisymptomatic, our study also provides a measurement of the prevalence of asymptomatic/paucisymptomatic SARS-CoV-2 infections. Our study suggests that essential workers have a 2-fold increase in viral exposure, when compared to non-essential workers that observed confinement. Additional serological surveys in different population subgroups will paint a broader picture of the effect of the confinement measures in the broader community.

16.
J Clin Virol ; 127: 104383, 2020 06.
Article in English | MEDLINE | ID: covidwho-1385847

ABSTRACT

BACKGROUND: Numerous nucleic acid amplification assays have recently received emergency use authorization (EUA) for the diagnosis of SARS-CoV-2 infection, and there is a need to assess their test performance relative to one another. OBJECTIVES: The aim of this study was to compare the test performance of the Hologic Panther Fusion SARS-CoV-2 assay targeting two regions of open reading frame 1ab (ORF1ab) to a high complexity molecular-based, laboratory-developed EUA from Stanford Health Care (SHC) targeting the SARS-CoV-2 envelope (E) gene. STUDY DESIGN: We performed a diagnostic comparison study by testing nasopharyngeal samples on the two assays. Assay agreement was assessed by overall percent agreement and Cohen's kappa coefficient. RESULTS: A total of 184 nasopharyngeal samples were tested using the two assays, of which 180 showed valid results and were included for the comparative analysis. Overall percent agreement between the assays was 98.3 % (95 % confidence interval (CI) 95.2-99.7) and kappa coefficient was 0.97 (95 % CI 0.93-1.0). One sample was detected on the SHC laboratory developed test (LDT) and not on the Panther Fusion, and had a Ct of 35.9. Conversely, 2 samples were detected on the Panther Fusion and not on the LDT, and had Ct values of 37.2 and 36.6. CONCLUSION: The Panther Fusion SARS-CoV-2 assay and the SHC LDT perform similarly on clinical nasopharyngeal swab specimens. Other considerations, including reagent availability, turnaround time, labor requirements, cost and instrument throughput should guide the decision of which assay to perform.


Subject(s)
Coronavirus Infections/diagnosis , Molecular Diagnostic Techniques/methods , Molecular Diagnostic Techniques/standards , Pneumonia, Viral/diagnosis , Reagent Kits, Diagnostic/standards , Viral Envelope Proteins/isolation & purification , Betacoronavirus/genetics , COVID-19 , Coronavirus Envelope Proteins , Humans , Nasopharynx/virology , Pandemics , Reproducibility of Results , SARS-CoV-2 , Viral Envelope Proteins/genetics
17.
J Med Virol ; 93(9): 5560-5567, 2021 Sep.
Article in English | MEDLINE | ID: covidwho-1363699

ABSTRACT

Quantitation of antibodies to the spike protein of severe acute respiratory syndrome coronavirus 2  (SARS-CoV-2) was performed for the detection of adaptive immune response in healthcare workers (HCWs) vaccinated with CorovaVac. We prospectively recruited HCWs from a university hospital in Turkey. Serum samples from 1072 HCWs were obtained following 28 days of the first, and 21 days of the second dose. Detection and quantitation of SARS-CoV-2 antispike antibodies were performed by the chemiluminescent microparticle immunoassay (SARS-CoV-2 IgG II Quant; Abbott). Results greater than or equal to the cutoff value 50.0 AU/ml were reported as positive. After the first dose, antispike antibodies were detected in 834 of 1072 (77.8%) HCWs. Seropositivity was higher among females (84.6%) than males (70.6%) (p < 0.001) and was found to be highest in both women and men between the ages of 18-34. After the second dose, antibodies were detected in 1008 of 1012 (99.6%) HCWs. Antibody titers were significantly higher in those who had coronavirus disease-2019 before vaccination than those who did not (p < 0.001). Antibody positivity and median antibody titers were significantly less in HCWs with chronic diseases compared to those without (p < 0.05 and p < 0.001, respectively). In conclusion, our findings indicated that a relatively high frequency (99.6%) of humoral immunity was produced in HCWs aged 18-59 after two doses of CoronaVac. Quantitation of antibodies may help facilitate longitudinal monitoring of the antibody response, which will be especially useful in deciding the dose of the vaccine in vulnerable groups such as those over 60 years of age and those with chronic diseases.


Subject(s)
Antibodies, Viral/blood , COVID-19 Vaccines/immunology , COVID-19/prevention & control , Immunoglobulin G/blood , Spike Glycoprotein, Coronavirus/immunology , Adolescent , Adult , Antibody Formation , COVID-19/immunology , Female , Health Personnel , Humans , Immunization Schedule , Male , Middle Aged , Prospective Studies , Turkey , Young Adult
18.
J Med Virol ; 93(9): 5538-5543, 2021 Sep.
Article in English | MEDLINE | ID: covidwho-1363694

ABSTRACT

In the current coronavirus disease 2019 (COVID-19) pandemic there is a mass screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) happening around the world due to the extensive spread of the infections. There is a high demand for rapid diagnostic tests to expedite the identification of cases and to facilitate early isolation and control spread. Hence this study evaluates six different rapid nucleic acid detection assays that are commercially available for SARS-CoV-2 virus detection. Nasopharyngeal samples were collected from 4981 participants and were tested for the SARS-CoV-2 virus by the gold standard real-time reverse-transcription polymerase chain reaction (RT-PCR) method and with one of these six rapid methods of detection. Evaluation of the rapid nucleic acid detection assays was done by comparing the results of these rapid methods with the gold standard RT-qPCR results for SARS-COV-2 detection. AQ-TOP had the highest sensitivity (98%) and a strong kappa value of 0.943 followed by Genechecker and Abbot ID NOW. The POCKIT (ii RT-PCR) assay had the highest test accuracy of 99.29% followed by Genechecker and Cobas Liat. Atila iAMP showed the highest percentage of invalid reports (35.5%) followed by AQ-TOP with 6% and POCKIT with 3.7% of invalid reports. Genechecker system, Abbott ID NOW, and Cobas Liat were found to have the best performance and agreement when compared with the standard RT-PCR for COVID-19 detection. With further research, these rapid tests have the potential to be employed in large-scale screening of COVID-19.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , SARS-CoV-2/isolation & purification , COVID-19 Nucleic Acid Testing/instrumentation , COVID-19 Nucleic Acid Testing/standards , Humans , Nasopharynx/virology , Real-Time Polymerase Chain Reaction , SARS-CoV-2/genetics , Sensitivity and Specificity , United Arab Emirates
19.
J Med Virol ; 93(9): 5333-5338, 2021 Sep.
Article in English | MEDLINE | ID: covidwho-1363672

ABSTRACT

The accurate laboratory detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a crucial element in the fight against coronavirus disease 2019 (COVID-19). Reverse transcription-polymerase chain reaction testing on combined oral and nasopharyngeal swab (ONPS) suffers from several limitations, including the need for qualified personnel, the discomfort caused by invasive nasopharyngeal sample collection, and the possibility of swab and transport media shortage. Testing on saliva would represent an advancement. The aim of this study was to compare the concordance between saliva samples and ONPS for the detection of SARS-CoV-2 on various commercial and laboratory-developed tests (LDT). Individuals were recruited from eight institutions in Quebec, Canada, if they had SARS-CoV-2 RNA detected on a recently collected ONPS, and accepted to provide another ONPS, paired with saliva. Assays available in the different laboratories (Abbott RealTime SARS-CoV-2, Cobas® SARS-CoV-2, Simplexa™ COVID-19 Direct, Allplex™ 2019-nCoV, RIDA®GENE SARS-CoV-2, and an LDT preceded by three different extraction methods) were used to determine the concordance between saliva and ONPS results. Overall, 320 tests were run from a total of 125 saliva and ONPS sample pairs. All assays yielded similar sensitivity when saliva was compared to ONPS, with the exception of one LDT (67% vs. 93%). The mean difference in cycle threshold (∆C t ) was generally (but not significantly) in favor of the ONPS for all nucleic acid amplification tests. The maximum mean ∆​​​​​C t was 2.0, while individual ∆C t varied importantly from -17.5 to 12.4. Saliva seems to be associated with sensitivity similar to ONPS for the detection of SARS-CoV-2 by various assays.


Subject(s)
COVID-19 Nucleic Acid Testing/standards , COVID-19/diagnosis , Diagnostic Tests, Routine/standards , RNA, Viral/genetics , SARS-CoV-2/genetics , COVID-19/epidemiology , COVID-19/virology , COVID-19 Nucleic Acid Testing/instrumentation , COVID-19 Nucleic Acid Testing/methods , Diagnostic Tests, Routine/instrumentation , Diagnostic Tests, Routine/methods , Humans , Mouth/virology , Nasopharynx/virology , Quebec/epidemiology , Saliva/virology , Sensitivity and Specificity , Specimen Handling/standards
20.
J Pediatric Infect Dis Soc ; 10(6): 706-713, 2021 Aug 14.
Article in English | MEDLINE | ID: covidwho-1358465

ABSTRACT

BACKGROUND: Recently, cases of multisystem inflammatory syndrome in children (MIS-C) associated with coronavirus disease 2019 (COVID-19) have been reported worldwide. Negative polymerase chain reaction (RT-PCR) testing associated with positive serology in most of the cases suggests a postinfectious syndrome. Because the pathophysiology of this syndrome is still poorly understood, extensive virological and immunological investigations are needed. METHODS: We report a series of 4 pediatric patients admitted to Geneva University Hospitals with persistent fever and laboratory evidence of inflammation meeting the published definition of MIS-C related to COVID-19, to whom an extensive virological and immunological workup was performed. RESULTS: RT-PCRs on multiple anatomical compartments were negative, whereas anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoglobulin A (IgA) and immunoglobulin G (IgG) were strongly positive by enzyme-linked immunosorbent assay and immunofluorescence. Both pseudoneutralization and full virus neutralization assays showed the presence of neutralizing antibodies in all children, confirming a recent infection with SARS-CoV-2. The analyses of cytokine profiles revealed an elevation in all cytokines, as reported in adults with severe COVID-19. Although differing in clinical presentation, some features of MIS-C show phenotypic overlap with hemophagocytic lymphohistiocytosis (HLH). In contrast to patients with primary HLH, our patients showed normal perforin expression and natural killer (NK) cell degranulation. The levels of soluble interleukin (IL)-2 receptor (sIL-2R) correlated with the severity of disease, reflecting recent T-cell activation. CONCLUSION: Our findings suggest that MIS-C related to COVID-19 is caused by a postinfectious inflammatory syndrome associated with an elevation in all cytokines, and markers of recent T-cell activation (sIL-2R) occurring despite a strong and specific humoral response to SARS-CoV-2. Further functional and genetic analyses are essential to better understand the mechanisms of host-pathogen interactions.


Subject(s)
COVID-19 , Antibodies, Neutralizing , Child , Humans , SARS-CoV-2 , Systemic Inflammatory Response Syndrome
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