A novel strategy for high-specificity, high-sensitivity, and high-throughput study for gut microbiome metabolism of aromatic carboxylic acids

2021 ◽  
Author(s):  
Ningning Zhao ◽  
Zhiqiang Liu ◽  
Junpeng Xing ◽  
Zhong Zheng ◽  
Fengrui Song ◽  
...  
Keyword(s):  
Carboxylic Acids ◽  
High Throughput ◽  
Gut Microbiome ◽  
High Sensitivity ◽  
High Specificity ◽  
Aromatic Carboxylic Acids ◽  
Novel Strategy ◽  
High Throughput Study

scholarly journals Performance and Implementation Evaluation of the Abbott BinaxNOW Rapid Antigen Test in a High-throughput Drive-through Community Testing Site in Massachusetts

2021 ◽  
Author(s):  
Nira R. Pollock ◽  
Jesica R. Jacobs ◽  
Kristine Tran ◽  
Amber Cranston ◽  
Sita Smith ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
High Throughput ◽  
Point Of Care ◽  
High Sensitivity ◽  
High Specificity ◽  
Molecular Testing ◽  
Rt Pcr ◽  
Asymptomatic Children ◽  
Testing Site ◽  
Very High

AbstractBackgroundRapid diagnostic tests (RDTs) for SARS-CoV-2 antigens (Ag) that can be performed at point-of-care (POC) can supplement molecular testing and help mitigate the COVID-19 pandemic. Deployment of an Ag RDT requires an understanding of its operational and performance characteristics under real-world conditions and in relevant subpopulations. We evaluated the Abbott BinaxNOW™ COVID-19 Ag Card in a high-throughput, drive-through, free community testing site in Massachusetts (MA) using anterior nasal (AN) swab RT-PCR for clinical testing.MethodsIndividuals presenting for molecular testing in two of seven lanes were offered the opportunity to also receive BinaxNOW testing. Dual AN swabs were collected from symptomatic and asymptomatic children (≤ 18 years) and adults. BinaxNOW testing was performed in a testing pod with temperature/humidity monitoring. One individual performed testing and official result reporting for each test, but most tests had a second independent reading to assess inter-operator agreement. Positive BinaxNOW results were scored as faint, medium, or strong. Positive BinaxNOW results were reported to patients by phone and they were instructed to isolate pending RT-PCR results. The paired RT-PCR result was the reference for sensitivity and specificity calculations.ResultsOf 2482 participants, 1380 adults and 928 children had paired RT-PCR/BinaxNOW results and complete symptom data. 974/1380 (71%) adults and 829/928 (89%) children were asymptomatic. BinaxNOW had 96.5% (95% confidence interval [CI] 90.0-99.3) sensitivity and 100% (98.6-100.0) specificity in adults within 7 days of symptoms, and 84.6% (65.1-95.6) sensitivity and 100% (94.5-100.0) specificity in children within 7 days of symptoms. Sensitivity and specificity in asymptomatic adults were 70.2% (56.6-81.6) and 99.6% (98.9-99.9), respectively, and in asymptomatic children were 65.4% (55.6-74.4) and 99.0% (98.0-99.6), respectively. By cycle threshold (Ct) value cutoff, sensitivity in all subgroups combined (n=292 RT-PCR-positive individuals) was 99.3% with Ct ≤25, 95.8% with ≤30, and 81.2% with ≤35. Twelve false positive BinaxNOW results (out of 2308 tests) were observed; in all twelve, the test bands were faint but otherwise normal, and were noted by both readers. One invalid BinaxNOW result was identified. Inter-operator agreement (positive versus negative BinaxNOW result) was 100% (n = 2230/2230 double reads). Each operator was able to process 20 RDTs per hour. In a separate set of 30 specimens (from individuals with symptoms ≤7 days) run at temperatures below the manufacturer’s recommended range (46-58.5°F), sensitivity was 66.7% and specificity 95.2%.ConclusionsBinaxNOW had very high specificity in both adults and children and very high sensitivity in newly symptomatic adults. Overall, 95.8% sensitivity was observed with Ct ≤ 30. These data support public health recommendations for use of the BinaxNOW test in adults with symptoms for ≤7 days without RT-PCR confirmation. Excellent inter-operator agreement indicates that an individual can perform and read the BinaxNOW test alone. A skilled laboratorian can perform and read 20 tests per hour. Careful attention to temperature is critical.

scholarly journals Performance Characteristics of Four High-Throughput Immunoassays for Detection of IgG Antibodies against SARS-CoV-2

2020 ◽  
Vol 58 (8) ◽  
Author(s):  
Elitza S. Theel ◽  
Julie Harring ◽  
Heather Hilgart ◽  
Dane Granger
Keyword(s):  
High Throughput ◽  
High Sensitivity ◽  
High Specificity ◽  
Cross Reactivity ◽  
Clinical Diagnostics ◽  
Igg Antibodies ◽  
Serum Samples ◽  
Predictive Values ◽  
Serologic Tests ◽  
Convalescent Phase

ABSTRACT The role of serologic testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in both the clinical and public health settings, will continue to evolve as we gain increasing insight into our immune response to the virus. Here, we evaluated four high-throughput serologic tests for detection of anti-SARS-CoV-2 IgG antibodies, from Abbott Laboratories (Abbott Park, IL), Epitope Diagnostics, Inc. (San Diego, CA), Euroimmun (Lubeck, Germany), and Ortho-Clinical Diagnostics (Rochester, NY), using a panel of serially collected serum samples (n = 224) from 56 patients with confirmed coronavirus disease 2019 (COVID-19), healthy donor sera from 2018, and a cross-reactivity serum panel collected in early 2020. The sensitivities of the Abbott, Epitope, Euroimmun, and Ortho-Clinical IgG assays in convalescent-phase serum samples collected more than 14 days post-symptom onset or post-initial positive reverse transcriptase PCR (RT-PCR) result were 92.9% (78/84), 88.1% (74/84), 97.6% (82/84), and 98.8% (83/84), respectively. Among unique convalescent patients, sensitivities of the Abbott, Epitope, Euroimmun, and Ortho-Clinical anti-SARS-CoV-2 IgG assays were 97.3% (36/37), 73% (27/37), 94.6% (35/37), and 97.3% (36/37), respectively. Overall assay specificity/positive predictive values based on a 5% prevalence rate were 99.6%/92.8%, 99.6%/90.6%, 98.0%/71.2%, and 99.6%/92.5%, respectively, for the Abbott, Epitope, Euroimmun, and Ortho-Clinical IgG assays. In conclusion, we show high sensitivity in convalescent-phase sera and high specificity for the Abbott, Euroimmun, and Ortho-Clinical anti-SARS-CoV-2 IgG assays. With the unprecedented influx of commercially available serologic tests for detection of antibodies against SARS-CoV-2, it remains imperative that laboratories thoroughly evaluate such assays for accuracy prior to implementation.

scholarly journals Laboratory and Clinical Evaluation of DNA Microarray for the Detection of Carbapenemase Genes in Gram-Negative Bacteria from Hospitalized Patients

2019 ◽  
Vol 2019 ◽  
pp. 1-13
Author(s):  
Yi Song ◽  
Fengna Dou ◽  
Sha He ◽  
Yu Zhou ◽  
Qiqi Liu
Keyword(s):  
Dna Microarray ◽  
High Throughput ◽  
Sanger Sequencing ◽  
Limit Of Detection ◽  
High Sensitivity ◽  
High Specificity ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Specificity And Sensitivity ◽  
Global Threat

Background. The prevalence of a variety of carbapenemases in Gram-negative bacteria (GNB) has posed a global threat on clinical control and management. Monitoring and controlling the carbapenemase-producing GNB became imperative tasks for many healthcare centers. The aim of this study was to develop a high-throughput, specific, sensitive, and rapid DNA microarray-based method for the diagnosis, phenotypic confirmation, and molecular epidemiological study of carbapenemase genes. Methods. We targeted a panel of eight carbapenemase genes, including blaKPC, blaNDM-1, blaOXA-23, blaOXA-48, blaOXA-51, blaIMP, blaVIM, and blaDIM for detection. Ultrasensitive chemiluminescence (CL) detection method was developed and used to simultaneously detect eight carbapenemase genes, and plasmids were established as positive or limit of detection (LOD) reference materials. Antibiotic susceptibility was determined by disk diffusion according to Clinical and Laboratory Standards Institute (CLSI) guidelines in order to screen clinical isolates resistant to carbapenem antibiotics as well as Sanger sequencing which was used to confirm the reliability of the results presented by DNA microarray. Results. Eight carbapenemase genes could be detected with high sensitivity and specificity. The absolute LOD of this strategy to detect serially diluted plasmids of eight carbapenemase genes was 102- 103copies/μL. Then, 416 specimens collected from hospital were detected and the results showed 96.6% concordance between the phenotypic and microarray tests. Compared with Sanger sequencing, a specificity and sensitivity of 100% were recorded for blaNDM-1, blaIMP, blaVIM, and blaDIM genes. The specificity for blaKPC, blaOXA-23, blaOXA-48, and blaOXA-51 genes was 100% and the sensitivity was 98.5%, 97.6%, 95.7%, and 97.9%, respectively. The overall consistency rate between the sequencing and microarray is 97.8%. Conclusions. The proposed ultrasensitive CL imaging DNA hybridization has high specificity, sensitivity, and reproducibility and could detect and differentiate clinical specimens that carried various carbapenemase genes, suggesting that the method can conveniently be customized for high-throughput detection of the carbapenemase-producing GNB and can be easily adapted for various clinical applications.

scholarly journals Performance and Implementation Evaluation of the Abbott BinaxNOW Rapid Antigen Test in a High-throughput Drive-through Community Testing Site in Massachusetts

2021 ◽  
Author(s):  
Nira R. Pollock ◽  
Jesica R. Jacobs ◽  
Kristine Tran ◽  
Amber E. Cranston ◽  
Sita Smith ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
High Throughput ◽  
Point Of Care ◽  
High Sensitivity ◽  
High Specificity ◽  
Molecular Testing ◽  
Rt Pcr ◽  
Asymptomatic Children ◽  
Testing Site ◽  
Very High

Background: Rapid diagnostic tests (RDTs) for SARS-CoV-2 antigens (Ag) that can be performed at point-of-care (POC) can supplement molecular testing and help mitigate the COVID-19 pandemic. Deployment of an Ag RDT requires an understanding of its operational and performance characteristics under real-world conditions and in relevant subpopulations. We evaluated the Abbott BinaxNOW™ COVID-19 Ag Card in a high-throughput, drive-through, free community testing site in Massachusetts (MA) using anterior nasal (AN) swab RT-PCR for clinical testing. Methods: Individuals presenting for molecular testing in two of seven lanes were offered the opportunity to also receive BinaxNOW testing. Dual AN swabs were collected from symptomatic and asymptomatic children (≤ 18 years) and adults. BinaxNOW testing was performed in a testing pod with temperature/humidity monitoring. One individual performed testing and official result reporting for each test, but most tests had a second independent reading to assess inter-operator agreement. Positive BinaxNOW results were scored as faint, medium, or strong. Positive BinaxNOW results were reported to patients by phone and they were instructed to isolate pending RT-PCR results. The paired RT-PCR result was the reference for sensitivity and specificity calculations. Results: Of 2482 participants, 1380 adults and 928 children had paired RT-PCR/BinaxNOW results and complete symptom data. 974/1380 (71%) adults and 829/928 (89%) children were asymptomatic. BinaxNOW had 96.5% (95% confidence interval [CI] 90.0- 99.3) sensitivity and 100% (98.6-100.0) specificity in adults within 7 days of symptoms, and 84.6% (65.1-95.6) sensitivity and 100% (94.5-100.0) specificity in children within 7 days of symptoms. Sensitivity and specificity in asymptomatic adults were 70.2% (56.6-81.6) and 99.6% (98.9-99.9), respectively, and in asymptomatic children were 65.4% (55.6-74.4) and 99.0% (98.0-99.6), respectively. By cycle threshold (Ct) value cutoff, sensitivity in all subgroups combined (n=292 RT-PCR-positive individuals) was 99.3% with Ct ≤25, 95.8% with ≤30, and 81.2% with ≤35. Twelve false positive BinaxNOW results (out of 2308 tests) were observed; in all twelve, the test bands were faint but otherwise normal, and were noted by both readers. One invalid BinaxNOW result was identified. Inter-operator agreement (positive versus negative BinaxNOW result) was 100% (n = 2230/2230 double reads). Each operator was able to process 20 RDTs per hour. In a separate set of 30 specimens (from individuals with symptoms ≤7 days) run at temperatures below the manufacturer’s recommended range (46-58.5°F), sensitivity was 66.7% and specificity 95.2%. Conclusions: BinaxNOW had very high specificity in both adults and children and very high sensitivity in newly symptomatic adults. Overall, 95.8% sensitivity was observed with Ct ≤ 30. These data support public health recommendations for use of the BinaxNOW test in adults with symptoms for ≤7 days without RT-PCR confirmation. Excellent inter-operator agreement indicates that an individual can perform and read the BinaxNOW test alone. A skilled laboratorian can perform and read 20 tests per hour. Careful attention to temperature is critical.

scholarly journals Optimized RNA-targeting CRISPR/Cas13d technology outperforms shRNA in identifying functional circRNAs

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yang Zhang ◽  
Tuan M. Nguyen ◽  
Xiao-Ou Zhang ◽  
Limei Wang ◽  
Tin Phan ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
High Throughput ◽  
Biological Effect ◽  
Human Hepatocellular Carcinoma ◽  
Guide Rnas ◽  
Rna Targeting ◽  
Short Hairpin ◽  
Short Hairpin Rnas ◽  
Screening Library ◽  
High Throughput Study

AbstractShort hairpin RNAs (shRNAs) are used to deplete circRNAs by targeting back-splicing junction (BSJ) sites. However, frequent discrepancies exist between shRNA-mediated circRNA knockdown and the corresponding biological effect, querying their robustness. By leveraging CRISPR/Cas13d tool and optimizing the strategy for designing single-guide RNAs against circRNA BSJ sites, we markedly enhance specificity of circRNA silencing. This specificity is validated in parallel screenings by shRNA and CRISPR/Cas13d libraries. Using a CRISPR/Cas13d screening library targeting > 2500 human hepatocellular carcinoma-related circRNAs, we subsequently identify a subset of sorafenib-resistant circRNAs. Thus, CRISPR/Cas13d represents an effective approach for high-throughput study of functional circRNAs.

scholarly journals From Multiplex Serology to Serolomics—A Novel Approach to the Antibody Response against the SARS-CoV-2 Proteome

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 749
Author(s):  
Julia Butt ◽  
Rajagopal Murugan ◽  
Theresa Hippchen ◽  
Sylvia Olberg ◽  
Monique van Straaten ◽  
...  
Keyword(s):  
Antibody Response ◽  
High Throughput ◽  
Large Population ◽  
High Sensitivity ◽  
Population Based ◽  
Antibody Responses ◽  
Hek293 Cells ◽  
Novel Approach ◽  
Assay Performance ◽  
Multiplex Serology

The emerging SARS-CoV-2 pandemic entails an urgent need for specific and sensitive high-throughput serological assays to assess SARS-CoV-2 epidemiology. We, therefore, aimed at developing a fluorescent-bead based SARS-CoV-2 multiplex serology assay for detection of antibody responses to the SARS-CoV-2 proteome. Proteins of the SARS-CoV-2 proteome and protein N of SARS-CoV-1 and common cold Coronaviruses (ccCoVs) were recombinantly expressed in E. coli or HEK293 cells. Assay performance was assessed in a COVID-19 case cohort (n = 48 hospitalized patients from Heidelberg) as well as n = 85 age- and sex-matched pre-pandemic controls from the ESTHER study. Assay validation included comparison with home-made immunofluorescence and commercial enzyme-linked immunosorbent (ELISA) assays. A sensitivity of 100% (95% CI: 86–100%) was achieved in COVID-19 patients 14 days post symptom onset with dual sero-positivity to SARS-CoV-2 N and the receptor-binding domain of the spike protein. The specificity obtained with this algorithm was 100% (95% CI: 96–100%). Antibody responses to ccCoVs N were abundantly high and did not correlate with those to SARS-CoV-2 N. Inclusion of additional SARS-CoV-2 proteins as well as separate assessment of immunoglobulin (Ig) classes M, A, and G allowed for explorative analyses regarding disease progression and course of antibody response. This newly developed SARS-CoV-2 multiplex serology assay achieved high sensitivity and specificity to determine SARS-CoV-2 sero-positivity. Its high throughput ability allows epidemiologic SARS-CoV-2 research in large population-based studies. Inclusion of additional pathogens into the panel as well as separate assessment of Ig isotypes will furthermore allow addressing research questions beyond SARS-CoV-2 sero-prevalence.

scholarly journals Radiochemistry, Production Processes, Labeling Methods, and ImmunoPET Imaging Pharmaceuticals of Iodine-124

Molecules ◽  
2021 ◽  
Vol 26 (2) ◽  
pp. 414
Author(s):  
Krishan Kumar ◽  
Arijit Ghosh
Keyword(s):  
Physical Properties ◽  
Imaging Modality ◽  
Target Selection ◽  
Positron Emission Tomography Imaging ◽  
High Sensitivity ◽  
High Specificity ◽  
Production Processes ◽  
Positron Emission ◽  
Significant Research ◽  
Clinical Environments

Target-specific biomolecules, monoclonal antibodies (mAb), proteins, and protein fragments are known to have high specificity and affinity for receptors associated with tumors and other pathological conditions. However, the large biomolecules have relatively intermediate to long circulation half-lives (>day) and tumor localization times. Combining superior target specificity of mAbs and high sensitivity and resolution of the PET (Positron Emission Tomography) imaging technique has created a paradigm-shifting imaging modality, ImmunoPET. In addition to metallic PET radionuclides, 124I is an attractive radionuclide for radiolabeling of mAbs as potential immunoPET imaging pharmaceuticals due to its physical properties (decay characteristics and half-life), easy and routine production by cyclotrons, and well-established methodologies for radioiodination. The objective of this report is to provide a comprehensive review of the physical properties of iodine and iodine radionuclides, production processes of 124I, various 124I-labeling methodologies for large biomolecules, mAbs, and the development of 124I-labeled immunoPET imaging pharmaceuticals for various cancer targets in preclinical and clinical environments. A summary of several production processes, including 123Te(d,n)124I, 124Te(d,2n)124I, 121Sb(α,n)124I, 123Sb(α,3n)124I, 123Sb(3He,2n)124I, natSb(α, xn)124I, natSb(3He,n)124I reactions, a detailed overview of the 124Te(p,n)124I reaction (including target selection, preparation, processing, and recovery of 124I), and a fully automated process that can be scaled up for GMP (Good Manufacturing Practices) production of large quantities of 124I is provided. Direct, using inorganic and organic oxidizing agents and enzyme catalysis, and indirect, using prosthetic groups, 124I-labeling techniques have been discussed. Significant research has been conducted, in more than the last two decades, in the development of 124I-labeled immunoPET imaging pharmaceuticals for target-specific cancer detection. Details of preclinical and clinical evaluations of the potential 124I-labeled immunoPET imaging pharmaceuticals are described here.

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