scholarly journals Critical Aspects Concerning the Development of a Pooling Approach for SARS-CoV-2 Diagnosis Using Large-Scale PCR Testing

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 902
Author(s):  
Daniel Cruceriu ◽  
Oana Baldasici ◽  
Loredana Balacescu ◽  
Stefana Gligor-Popa ◽  
Mirela Flonta ◽  
...  
Keyword(s):  
Large Scale ◽  
Rna Extraction ◽  
Diagnostic Assay ◽  
Assay Sensitivity ◽  
Infected People ◽  
Multiple Parameters ◽  
Accurate Procedure ◽  
Manual Methods ◽  
Pooling Strategy ◽  
Critical Aspects

The primary approach to controlling the spread of the pandemic SARS-CoV-2 is to diagnose and isolate the infected people quickly. Our paper aimed to investigate the efficiency and the reliability of a hierarchical pooling approach for large-scale PCR testing for SARS-CoV-2 diagnosis. To identify the best conditions for the pooling approach for SARS-CoV-2 diagnosis by RT-qPCR, we investigated four manual methods for both RNA extraction and PCR assessment targeting one or more of the RdRp, N, S, and ORF1a genes, by using two PCR devices and an automated flux for SARS-CoV-2 detection. We determined the most efficient and accurate diagnostic assay, taking into account multiple parameters. The optimal pool size calculation included the prevalence of SARS-CoV-2, the assay sensitivity of 95%, an assay specificity of 100%, and a range of pool sizes of 5 to 15 samples. Our investigation revealed that the most efficient and accurate procedure for detecting the SARS-CoV-2 has a detection limit of 2.5 copies/PCR reaction. This pooling approach proved to be efficient and accurate in detecting SARS-CoV-2 for all samples with individual quantification cycle (Cq) values lower than 35, accounting for more than 94% of all positive specimens. Our data could serve as a comprehensive practical guide for SARS-CoV-2 diagnostic centers planning to address such a pooling strategy.

Open Source Software Implementation of Anatomical Segmentation

2018 ◽  
Author(s):  
Maggie Hess
Keyword(s):  
Large Scale ◽  
Focused Ultrasound ◽  
Region Growing ◽  
Ground Truth ◽  
Clot Lysis ◽  
Focused Ultrasound Surgery ◽  
Manual Methods ◽  
Areas Of Interest ◽  
Seed Points ◽  
Viable Method

Purpose: Intraventricular hemorrhage (IVH) affects nearly 15% of preterm infants. It can lead to ventricular dilation and cognitive impairment. To ablate IVH clots, MR-guided focused ultrasound surgery (MRgFUS) is investigated. This procedure requires accurate, fast and consistent quantification of ventricle and clot volumes. Methods: We developed a semi-autonomous segmentation (SAS) algorithm for measuring changes in the ventricle and clot volumes. Images are normalized, and then ventricle and clot masks are registered to the images. Voxels of the registered masks and voxels obtained by thresholding the normalized images are used as seed points for competitive region growing, which provides the final segmentation. The user selects the areas of interest for correspondence after thresholding and these selections are the final seeds for region growing. SAS was evaluated on an IVH porcine model.  Results: SAS was compared to ground truth manual segmentation (MS) for accuracy, efficiency, and consistency. Accuracy was determined by comparing clot and ventricle volumes produced by SAS and MS. In Two-One-Sided Test, SAS and MS were found to be significantly equivalent (p < 0.01). SAS on average was found to be 15 times faster than MS (p < 0.01). Consistency was determined by repeated segmentation of the same image by both SAS and manual methods, SAS being significantly more consistent than MS (p < 0.05).  Conclusion: SAS is a viable method to quantify the IVH clot and the lateral brain ventricles and it is serving in a large- scale porcine study of MRgFUS treatment of IVH clot lysis.

scholarly journals RT-dPCR in Mosquito Samples for ZIKV Detection: Effects of RNA Extraction and Reverse Transcription in Target Concentration

Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 827
Author(s):  
Paula Rodrigues de Almeida ◽  
Ana Karolina Antunes Eisen ◽  
Meriane Demoliner ◽  
Fernando Rosado Spilki
Keyword(s):  
Reverse Transcription ◽  
Tissue Concentration ◽  
Critical Role ◽  
Rna Extraction ◽  
High Sensitivity ◽  
Clinical Samples ◽  
Assay Sensitivity ◽  
Robust Detection ◽  
Detection Techniques ◽  
Mosquito Pool

Zika virus (ZIKV) is an important arbovirus, responsible for recent outbreaks of Guillain Barré Syndrome and Congenital Zika Syndrome (CZS). After thousands of CZS cases, ZIKV is under constant surveillance in Brazil. Reliable and robust detection techniques are required to minimize the influence of host inhibitors from clinical samples and mosquito pool samples. Reverse transcription Digital Polymerase Chain Reaction (RT-dPCR) is a technique that allows the accurate quantification of DNA targets with high sensitivity, and it is usually less affected by inhibitors than RT-qPCR. This study aimed to assess the influence of mosquito tissue, RNA extraction and cDNA synthesis in ZIKV PCR detection. Samples containing 0, 3 and 10 mosquitoes were spiked with ZIKV MR766 and serially diluted prior to RNA extraction and RT-dPCR for ZIKV. Two reverse transcription protocols were tested. Assay sensitivity allowed the detection of 1.197 copies/µL. A higher correlation between dilution factor and target quantification was observed in 10 mosquito pool samples. The lower quantification in samples diluted without mosquitoes highlights the critical role of the reverse transcription step in RNA detection, since it could be attributed to reverse transcriptase variable performance in samples with low overall RNA concentration. The results in mosquito pools indicate that mosquito tissues do not inhibit ZIKV RT-dPCR, and the RT-dPCR technique has good sensitivity and robustness for ZIKV detection in mosquito pool samples regardless of mosquito tissue concentration.

scholarly journals The Light and Shadow of Rapid Serological Tests for SARS-CoV-2 Infection: Results from a Study in a Large Emergency Department

2020 ◽  
Vol 17 (18) ◽  
pp. 6493
Author(s):  
Daniela Loconsole ◽  
Francesca Centrone ◽  
Caterina Morcavallo ◽  
Silvia Campanella ◽  
Anna Sallustio ◽  
...  
Keyword(s):  
Emergency Department ◽  
Real Time ◽  
Real Time Pcr ◽  
Respiratory Symptoms ◽  
Large Scale ◽  
Serological Test ◽  
Serological Tests ◽  
Serological Assay ◽  
Infected People ◽  
Screening Programs

A critical point in the management of the SARS-CoV-2 pandemic is the need to promptly identify the greatest number of infected people and to implement strict public health measures. In this study, the performance of a rapid serological test in a clinical setting was evaluated. Samples from 819 consecutive patients (with or without respiratory symptoms) admitted to a large Emergency Department were tested between 23 March and 21 April 2020. Patient samples were tested in a real-time PCR assay and a serological assay. In total, 148/819 patients (18.1%) tested positive for SARS-CoV-2 by real-time PCR. The serological test revealed that 70/819 patients (8.5%) had anti-SARS-CoV-2 IgM and/or IgG. The prevalence of anti-SARS-CoV-2 antibodies was significantly higher in patients with respiratory symptoms lasting for >7 days than in those with respiratory symptoms lasting for 0–7 days (p < 0.001). The serological assay had an overall sensitivity of 35.1% and an overall specificity of 97.3%. A high negative predictive value (96.7%) was reported for patients without respiratory symptoms. The results confirm that rapid serological assays alone are not sufficient for diagnosis of SARS-CoV-2 infection but can be incorporated into large-scale screening programs during periods in which the virus circulation is low.

scholarly journals A comprehensive RNA handling and transcriptomics guide for high-throughput processing of Plasmodium blood-stage samples

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Michal Kucharski ◽  
Jaishree Tripathi ◽  
Sourav Nayak ◽  
Lei Zhu ◽  
Grennady Wirjanata ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Rna Extraction ◽  
Transcriptome Data ◽  
High Quality ◽  
Diverse Range ◽  
Human Haemoglobin ◽  
Infected Blood ◽  
Blood Samples ◽  
Total Rna

Abstract Background Sequencing technology advancements opened new opportunities to use transcriptomics for studying malaria pathology and epidemiology. Even though in recent years the study of whole parasite transcriptome proved to be essential in understanding parasite biology there is no compiled up-to-date reference protocol for the efficient generation of transcriptome data from growing number of samples. Here, a comprehensive methodology on how to preserve, extract, amplify, and sequence full-length mRNA transcripts from Plasmodium-infected blood samples is presented that can be fully streamlined for high-throughput studies. Results The utility of various commercially available RNA-preserving reagents in a range of storage conditions was evaluated. Similarly, several RNA extraction protocols were compared and the one most suitable method for the extraction of high-quality total RNA from low-parasitaemia and low-volume blood samples was established. Furthermore, the criteria needed to evaluate the quality and integrity of Plasmodium RNA in the presence of human RNA was updated. Optimization of SMART-seq2 amplification method to better suit AT-rich Plasmodium falciparum RNA samples allowed us to generate high-quality transcriptomes from as little as 10 ng of total RNA and a lower parasitaemia limit of 0.05%. Finally, a modified method for depletion of unwanted human haemoglobin transcripts using in vitro CRISPR-Cas9 treatment was designed, thus improving parasite transcriptome coverage in low parasitaemia samples. To prove the functionality of the pipeline for both laboratory and field strains, the highest  2-hour resolution RNA-seq transcriptome for P. falciparum 3D7 intraerythrocytic life cycle available to  date was generated, and the entire protocol was applied to create the largest transcriptome data from Southeast Asian field isolates. Conclusions Overall, the presented methodology is an inclusive pipeline for generation of good quality transcriptomic data from a diverse range of Plasmodium-infected blood samples with varying parasitaemia and RNA inputs. The flexibility of this pipeline to be adapted to robotic handling will facilitate both small and large-scale future transcriptomic studies in the field of malaria.

scholarly journals SARS-CoV-2 RNA Extraction Using Magnetic Beads for Rapid Large-Scale Testing by RT-qPCR and RT-LAMP

Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 863 ◽  
Author(s):  
Steffen Klein ◽  
Thorsten G. Müller ◽  
Dina Khalid ◽  
Vera Sonntag-Buck ◽  
Anke-Mareil Heuser ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Large Scale ◽  
Rna Extraction ◽  
Magnetic Bead ◽  
Viral Rna ◽  
Detection Sensitivity ◽  
Detection Methods ◽  
N Gene ◽  
Extraction Protocol ◽  
Large Scale Testing

Rapid large-scale testing is essential for controlling the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The standard diagnostic pipeline for testing SARS-CoV-2 presence in patients with an ongoing infection is predominantly based on pharyngeal swabs, from which the viral RNA is extracted using commercial kits, followed by reverse transcription and quantitative PCR detection. As a result of the large demand for testing, commercial RNA extraction kits may be limited and, alternatively, non-commercial protocols are needed. Here, we provide a magnetic bead RNA extraction protocol that is predominantly based on in-house made reagents and is performed in 96-well plates supporting large-scale testing. Magnetic bead RNA extraction was benchmarked against the commercial QIAcube extraction platform. Comparable viral RNA detection sensitivity and specificity were obtained by fluorescent and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) using a primer set targeting the N gene, as well as RT-qPCR using a primer set targeting the E gene, showing that the RNA extraction protocol presented here can be combined with a variety of detection methods at high throughput. Importantly, the presented diagnostic workflow can be quickly set up in a laboratory without access to an automated pipetting robot.

Organizational Image and Identity Management in Large-Scale Sporting Events

2007 ◽  
Vol 21 (1) ◽  
pp. 15-40 ◽  
Author(s):  
Milena M. Parent ◽  
Peter O. Foreman
Keyword(s):  
Large Scale ◽  
Sport Management ◽  
Identity Management ◽  
Sporting Events ◽  
Sport Organizations ◽  
Organizational Image ◽  
Symbolic Communication ◽  
Stakeholder Groups ◽  
The Media ◽  
Critical Aspects

Although identity, image, and reputation are important issues for the sport management field, little research has examined how sport organizations construct and manage such intangible yet critical aspects of their organizations. This article addresses this gap in the literature by exploring the process of identity construction within organizing committees of major sporting events. The insights gained from two case studies indicate that committees draw on three types of identity referents: the nature of the event, context, and key individuals of organizing committees. These referents are projected as images from the organizing committee to various stakeholder groups and then reflected back to the organizing committee. In addition, images are often received by stakeholders through indirect channels of transmission, especially the media, further complicating the process of image and identity management. Finally, organizing committees attempt to manage the process primarily via verbal and symbolic communication strategies.

scholarly journals Pooling of samples to optimize SARS-CoV-2 diagnosis by RT-qPCR: comparative analysis of two protocols.

2020 ◽  
Author(s):  
Fabiana Volpato ◽  
Daiana Lima-Morales ◽  
Priscila Lamb Wink ◽  
Julia Willig ◽  
Fernanda de-Paris ◽  
...  
Keyword(s):  
Large Scale ◽  
Diagnostic Yield ◽  
Rna Extraction ◽  
Clinical Samples ◽  
The Novel ◽  
Positive Individual ◽  
Sample Pooling ◽  
Novel Coronavirus ◽  
The Individual ◽  
Pooled Samples

RT-qPCR for SARS-CoV-2 is the main diagnostic test used to identify the novel coronavirus. Several countries have used large scale SARS-CoV-2 RT-qPCR testing as one of the important strategies for combating the pandemic. In order to process the massive needs for coronavirus testing, the usual throughput of routine clinical laboratories has reached and often surpassed its limits and new approaches to cope with this challenge must be developed. This study has aimed to evaluate the use pool of samples as a strategy to optimize the diagnostic of SARS-CoV-2 by RT-qPCR in a general population. A total of 220 naso/orofaryngeal swab samples were collected and tested using two different protocols of sample pooling. In the first protocol (Protocol A); 10 clinical samples were pooled before RNA extraction. The second protocol (Protocol B) consisted of pooling the already extracted RNAs from 10 individual samples. Results from Protocol A were identical (100% agreement) with the individual results. However, for results from Protocol B, reduced agreement (91%) was observed in relation to results obtained by individual testing. Inconsistencies observed were related to RT-qPCR results with higher Cycle Thresholds (Ct > 32.73). Furthermore, in pools containing more than one positive individual, the Ct of the pool was equivalent to the lowest Ct among the individual results. These results provide additional evidence in favor of the clinical use of pooled samples for SARS-CoV-2 diagnosis by RT-qPCR and suggest that pooling of samples before RNA extraction is preferrable in terms of diagnostic yield.

scholarly journals Oil Immersed Lossless Total Analysis System (OIL-TAS): Integrated RNA Extraction and Detection for SARS-CoV-2 Testing

2020 ◽  
Author(s):  
Duane S. Juang ◽  
Terry D. Juang ◽  
Dawn M. Dudley ◽  
Christina M. Newman ◽  
Thomas C. Friedrich ◽  
...  
Keyword(s):  
Infectious Disease ◽  
Large Scale ◽  
Rna Extraction ◽  
Cost Effective ◽  
Acid Extraction ◽  
Input Concentration ◽  
Total Analysis System ◽  
Total Analysis ◽  
Analysis System ◽  
Disease Testing

AbstractThe coronavirus disease 2019 (COVID-19) pandemic exposed difficulties in scaling current quantitative PCR (qPCR)-based diagnostic methodologies for large-scale infectious disease testing. Bottlenecks include the lengthy multi-step process of nucleic acid extraction followed by qPCR readouts, which require costly instrumentation and infrastructure, as well as reagent and plastic consumable shortages stemming from supply chain constraints. Here we report a novel Oil Immersed Lossless Total Analysis System (OIL-TAS), which integrates RNA extraction and detection onto a single device that is simple, rapid, cost effective, uses minimal supplies and requires reduced infrastructure to perform. We validated the performance of OIL-TAS using contrived samples containing inactivated SARS-CoV-2 viral particles, which show that the assay can reliably detect an input concentration of 10 copies/μL and sporadically detect down to 1 copy/μL. The OIL-TAS method can serve as a faster, cheaper, and easier-to-deploy alternative to current qPCR-based methods for infectious disease testing.

scholarly journals Educational spaces and environments: in times of Covid-19 pandemic

2021 ◽  
Vol 5 (6) ◽  
pp. 193-196
Author(s):  
Jailma Cruz da Silva ◽  
Orliel dos Santos de Jesus
Keyword(s):  
Teaching And Learning ◽  
Large Scale ◽  
Educational Programs ◽  
Infected People ◽  
Knowing That ◽  
Practical Education ◽  
Educational Spaces ◽  
The World ◽  
The Impact ◽  
Different Levels

The appearance of COVID-19 brought to Brazil, and to the world, innumerable methods to contain the increase of infected people. These methods are necessary to avoid the spread of the virus and include social distancing and quarantine of the population. Knowing that these methods have a big impact on the educational system. This study has as its objective to verify the process of teaching and learning, in educational spaces and environments, in times of a pandemic. In order to look for a pattern in established actions in educational programs to incorporate in large scale the tools of educational technology at a distance (distance learning), for example platforms and virtual teaching environments designed to guarantee the pedagogical processes of learning and ameliorate the impact of absence from the classroom during the pandemic, and its post-pandemic consequences. We emphasize the importance of the teacher as mediator, understanding that this professional should respect the different levels of learning of the students and try to carry out activities that help with the improvement of practical education for the appropriate environment and spaces of interaction.

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