scholarly journals Saving Resources: SARS-CoV-2 Diagnostics by Real-Time RT-PCR Using Reduced Reaction Volumes

Diseases ◽  
2021 ◽  
Vol 9 (4) ◽  
pp. 84
Author(s):  
Sabine Bock ◽  
Bernd Hoffmann ◽  
Martin Beer ◽  
Kerstin Wernike
Keyword(s):  
Real Time ◽  
Viral Genome ◽  
Virus Detection ◽  
World Health ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Reaction Volume ◽  
Reaction Volumes ◽  
Health Organization ◽  
The Individual

Since the beginning of 2020, the betacoronavirus SARS-CoV-2 is causing a global pandemic of an acute respiratory disease termed COVID-19. The diagnostics of the novel disease is primarily based on direct virus detection by RT-PCR; however, the availability of test kits may become a major bottleneck, when millions of tests are performed per week. To increase the flexibility of SARS-CoV-2 diagnostics, three real-time RT-PCR assays listed on the homepage of the World Health Organization were selected and investigated regarding their compatibility with three different RT-PCR kits. Furthermore, the reaction volume of the PCR chemistry was reduced up to half of the original protocol to make the individual reactions more cost- and resource-effective. When testing dilution series of culture-grown virus, nearly identical quantification cycle values (Cq) were obtained for all RT-PCR assay/chemistry combinations. Regarding the SARS-CoV-2 detection in clinical samples, agreeing results were obtained for all combinations for virus negative specimens and swabs containing high to medium viral genome loads. In cases of very low SARS-CoV-2 genome loads (Cq > 36), inconsistent results were observed, with some test runs scoring negative and some positive. However, no preference of a specific target within the viral genome (E, RdRp, or N) or of a certain chemistry was seen. In summary, a reduction of the reaction volume and the type of PCR chemistry did not influence the PCR sensitivity.

scholarly journals Fast and low-cost detection of SARS-CoV-2 peptides by tandem mass spectrometry in clinical samples

2020 ◽  
Author(s):  
Karina Helena Morais Cardozo ◽  
Adriana Lebkuchen ◽  
Guilherme Goncalves Okai ◽  
Rodrigo Andrade Schuch ◽  
Luciana Godoy Viana ◽  
...  
Keyword(s):  
Real Time ◽  
Large Scale ◽  
Low Cost ◽  
Large Population ◽  
Virus Detection ◽  
Standard Test ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Fast Processing

Abstract The current outbreak of severe acute respiratory syndrome associated with coronavirus 2 (SARS-CoV-2) is pressing public health systems around the world, and large population testing is a key step to control this pandemic disease. Real-time reverse-transcription PCR (real-time RT-PCR) is the gold standard test for virus detection but the soaring demand for this test resulted in shortage of reagents and instruments, severely limiting its applicability to large-scale screening. To be used either as an alternative, or as a complement, to real-time RT-PCR testing, we developed a high-throughput targeted proteomics assay to detect SARS-CoV-2 proteins directly from clinical respiratory tract samples. Sample preparation was fully automated by using a modified magnetic particle-based proteomics approach implemented on a robotic liquid handler, enabling a fast processing of samples. The use of turbulent flow chromatography included four times multiplexed on-line sample cleanup and UPLC separation. MS/MS detection of three peptides from SARS-CoV-2 nucleoprotein and a 15N-labeled internal global standard was achieved within 2.5 min, enabling the analysis of more than 500 samples per day. The method was validated using 562 specimens previously analyzed by real-time RT-PCR and was able to detect over 83% of positive cases. No interference was found with samples from common respiratory viruses, including other coronaviruses (NL63, OC43, HKU1, and 229E). The strategy here presented has high sample stability and low cost and should be considered as an option to large population testing.

scholarly journals Real-time RT-PCR diagnostics of virus causing COVID-19

2020 ◽  
Vol 13 (1) ◽  
pp. 52-63 ◽  
Author(s):  
E. V. Goncharova ◽  
A. E. Donnikov ◽  
V. V. Kadochnikova ◽  
S. A. Morozova ◽  
M. N. Boldyreva ◽  
...  
Keyword(s):  
Real Time ◽  
Sensitivity And Specificity ◽  
High Sensitivity ◽  
Medical Product ◽  
World Health ◽  
Differential Diagnostics ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Study Results ◽  
Pcr Diagnostics

Aim: the study was aimed to develop a reagent kit for the real-time RT-PCR diagnostics of virus causing COVID-19.Materials and Methods. Three target sites were chosen in the genome SARS-CoV-2. The testing included 220 samples, 48 artificially created positive samples (made from patients’ biomaterial) and 172 clinical samples (scrapes from nasal and pharyngeal cavities, bronchoalveolar lavage, expectoration, endotracheal/nasopharyngeal aspirate, feces, post-mortem material), obtained from two medical centers. Preliminary, the obtained biomaterial was analyzed with a reagent kit of comparison. The evaluation was performed with a confidential interval CI 95%. The calculation of CI for the sensitivity and specificity was made based on the distribution of χ2.Results. The authors developed a technology of novel coronavirus infection (COVID-19) real-time RT-PCR diagnostics for the application in practical healthcare and proposed the variants of testing at all the stages (preanalytical, analytical, and post-analytical, including automated results processing). The proposed reagent kit meets the requirements of the World Health Organization and the Ministry of Healthcare of the Russian Federation. The study results demonstrated high sensitivity and specificity. The sensitivity was 100% (95% CI) 95.6–100%; the specificity was 100% (95% CI) 96.7–100%.Conclusion. The proposed reagent kit was registered in the RF as a medical product; the registration certificate No. RZN 2020/9948 dated 01.04.2020. The application of the reagent kit in network laboratories will provide patients with access to testing for the virus causing COVID-19 and contribute to quick differential diagnostics, improvement of pandemic control, and accurate statistics on the spread of the virus. 

scholarly journals Laboratory preparedness in EU/EEA countries for detection of novel avian influenza A(H7N9) virus, May 2013

2014 ◽  
Vol 19 (4) ◽  
Author(s):  
E Broberg ◽  
D Pereyaslov ◽  
M Struelens ◽  
D Palm ◽  
A Meijer ◽  
...  
Keyword(s):  
Avian Influenza ◽  
Real Time ◽  
Influenza A Virus ◽  
Influenza A ◽  
Virus Detection ◽  
Influenza Viruses ◽  
World Health ◽  
Reference Laboratory ◽  
The Novel ◽  
Rt Pcr

Following human infections with novel avian influenza A(H7N9) viruses in China, the European Centre for Disease Prevention and Control, the World Health Organization (WHO) Regional Office for Europe and the European Reference Laboratory Network for Human Influenza (ERLI-Net) rapidly posted relevant information, including real-time RT-PCR protocols. An influenza RNA sequence-based computational assessment of detection capabilities for this virus was conducted in 32 national influenza reference laboratories in 29 countries, mostly WHO National Influenza Centres participating in the WHO Global Influenza Surveillance and Response System (GISRS). Twenty-seven countries considered their generic influenza A virus detection assay to be appropriate for the novel A(H7N9) viruses. Twenty-two countries reported having containment facilities suitable for its isolation and propagation. Laboratories in 27 countries had applied specific H7 real-time RT-PCR assays and 20 countries had N9 assays in place. Positive control virus RNA was provided by the WHO Collaborating Centre in London to 34 laboratories in 22 countries to allow evaluation of their assays. Performance of the generic influenza A virus detection and H7 and N9 subtyping assays was good in 24 laboratories in 19 countries. The survey showed that ERLI-Net laboratories had rapidly developed and verified good capability to detect the novel A(H7N9) influenza viruses.

scholarly journals Fast and low-cost detection of SARS-CoV-2 peptides by tandem mass spectrometry in clinical samples

2020 ◽  
Author(s):  
Karina Helena Morais Cardozo ◽  
Adriana Lebkuchen ◽  
Guilherme Goncalves Okai ◽  
Rodrigo Andrade Schuch ◽  
Luciana Godoy Viana ◽  
...  
Keyword(s):  
Real Time ◽  
Large Scale ◽  
Low Cost ◽  
Large Population ◽  
Virus Detection ◽  
Standard Test ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Fast Processing

Abstract The current outbreak of severe acute respiratory syndrome associated with coronavirus 2 (SARS-CoV-2) is pressing public health systems around the world, and large population testing is a key step to control this pandemic disease. Real-time reverse-transcription PCR (real-time RT-PCR) is the gold standard test for virus detection but the soaring demand for this test resulted in shortage of reagents and instruments, severely limiting its applicability to large-scale screening. To be used either as an alternative, or as a complement, to real-time RT-PCR testing, we developed a high-throughput targeted proteomics assay to detect SARS-CoV-2 proteins directly from clinical respiratory tract samples. Sample preparation was fully automated by using a modified magnetic particle-based proteomics approach implemented on a robotic liquid handler, enabling a fast processing of samples. The use of turbulent flow chromatography included four times multiplexed on-line sample cleanup and UPLC separation. MS/MS detection of three peptides from SARS-CoV-2 nucleoprotein and a 15N-labeled internal global standard was achieved within 2.5 min, enabling the analysis of more than 500 samples per day. The method was validated using 562 specimens previously analyzed by real-time RT-PCR and was able to detect over 83% of positive cases. No interference was found with samples from common respiratory viruses, including other coronaviruses (NL63, OC43, HKU1, and 229E). The strategy here presented has high sample stability and low cost and should be considered as an option to large population testing.

scholarly journals Reliability of real-time RT-PCR tests to detect SARS-Cov-2: A literature review

2020 ◽  
Vol 11 ◽  
pp. 13 ◽  
Author(s):  
Clément Bezier ◽  
Géraldine Anthoine ◽  
Abdérafi Charki
Keyword(s):  
Real Time ◽  
World Health ◽  
Detection Methods ◽  
Rt Pcr ◽  
Health Crisis ◽  
The Face ◽  
Commercial Kits ◽  
Health Organization ◽  
Technical Capacity ◽  
The Impact

In the face of the COVID-19 (Coronavirus Disease 2019) pandemic, the World Health Organization (WHO) has urged countries to test the population more widely. Clinical laboratories have been confronted with a huge demand for testing and have had to make urgent preparations for staff training, to establish new analytical processes, reorganize the workspace, and stock up on specific equipment and diagnostic test kits. The reliability of SARS-Cov-2 test results is of critical importance, given the impact it has on patient care and the management of the health crisis. A review of the literature available for the period leading up to and including June 2020 on the reliability of SARS-Cov-2 (Severe Acute Respiratory Syndrome Coronavirus) detection methods using real-time RT PCR (Reverse Transcription - Polymerase Chain Reaction) brings together the primary factors teams of scientists claim or demonstrate to affect the reliability of results. A description is given of the RT-PCR testing method, followed by a presentation of the characteristics and validation techniques used. A summary of data from the literature on the reliability of tests and commercial kits for SARS-Cov-2 detection, including current uncertainties with regard to the molecular targets selected and genetic diversity of SARS-Cov-2 is provided. The limitations and perspectives are then discussed in detail in the light of the bibliographic data available. Many questions have been asked that still remain unanswered. The lack of knowledge about this novel virus, which appeared at the end of 2019, has a significant impact on the technical capacity to develop reliable, rapid and practical tools for its detection.

scholarly journals A Peptide-based Magnetic Chemiluminescence Enzyme Immunoassay for Serological Diagnosis of Corona Virus Disease 2019 (COVID-19)

2020 ◽  
Author(s):  
Xue-fei Cai ◽  
Juan Chen ◽  
Jie-li Hu ◽  
Quan-xin Long ◽  
Hai-jun Deng ◽  
...  
Keyword(s):  
Real Time ◽  
Virus Disease ◽  
Rna Extraction ◽  
Clinical Manifestations ◽  
Respiratory Illness ◽  
World Health ◽  
Sampling Location ◽  
Rt Pcr ◽  
Novel Coronavirus ◽  
Health Organization

AbstractA respiratory illness has been spreading rapidly in China, since its outbreak in Wuhan city, Hubei province in December 2019. The illness was caused by a novel coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Clinical manifestations related to SARS-CoV-2 infection ranged from no symptom to fatal pneumonia. World Health Organization (WHO) named the diseases associated with SARS-CoV-2 infection as COVID-19. Real time RT-PCR is the only laboratory test available till now to confirm the infection. However, the accuracy of real time RT-PCR depends on many factors, including sampling location and of methods, quality of RNA extraction and training of operators etc.. Variations in these factors might significantly lower the sensitivity of the detection. We developed a peptide-based luminescent immunoassay to detect IgG and IgM. Cut-off value of this assay was determined by the detection of 200 healthy sera and 167 sera from patients infected with other pathogens than SARS-CoV-2. To evaluate the performance of this assay, we detected IgG and IgM in the 276 sera from confirmed patients. The positive rate of IgG and IgM were 71.4% (197/276) and 57.2% (158/276) respectively. By combining with real time RT-PCR detection, this assay might help to enhance the accuracy of diagnosis of SARS-CoV-2 infection.

The relevance of dietetic approach of Ayurveda from the perspective of COVID-19 pandemic

2020 ◽  
Vol 11 (SPL1) ◽  
pp. 748-752
Author(s):  
Swapnali Khabade ◽  
Bharat Rathi ◽  
Renu Rathi
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Healthy Person ◽  
World Health ◽  
Physical And Mental Health ◽  
Leading Role ◽  
Global Pandemic ◽  
The People ◽  
The World ◽  
Health Organization ◽  
The Individual

A novel, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causes severe acute respiratory syndrome and spread globally from Wuhan, China. In March 2020 the World Health Organization declared the SARS-Cov-2 virus as a COVID- 19, a global pandemic. This pandemic happened to be followed by some restrictions, and specially lockdown playing the leading role for the people to get disassociated with their personal and social schedules. And now the food is the most necessary thing to take care of. It seems the new challenge for the individual is self-isolation to maintain themselves on the health basis and fight against the pandemic situation by boosting their immunity. Food organised by proper diet may maintain the physical and mental health of the individual. Ayurveda aims to promote and preserve the health, strength and the longevity of the healthy person and to cure the disease by properly channelling with and without Ahara. In Ayurveda, diet (Ahara) is considered as one of the critical pillars of life, and Langhana plays an important role too. This article will review the relevance of dietetic approach described in Ayurveda with and without food (Asthavidhi visheshaytana & Lanhgan) during COVID-19 like a pandemic.

Perancangan Sistem Informasi “DIAMONS” (Diabetes Monitoring System) Berbasis Internet of Things (IoT)

2019 ◽  
Vol 6 (1) ◽  
pp. 39-51
Author(s):  
Endang Sri Rahayu ◽  
Nurul Amalia
Keyword(s):  
Internet Of Things ◽  
World Health Organization ◽  
Real Time ◽  
Monitoring System ◽  
World Health ◽  
Health Organization ◽  
Diabetes Monitoring

Diabetes merupakan penyakit “silent killer” yang ditandai dengan peningkatan kadar glukosa darahdan kegagalan sekresi insulin. World Health Organization (WHO) pada tahun 2016 menyatakanbahwa diabetes menduduki urutan ke-6 sebagai penyakit mematikan di Indonesia. Sehingga upayapencegahan dan penanganan diabetes perlu mendapat perhatian yang serius. Internet of Things (IoT)dapat dijadikan sarana penunjang dalam penanganan penyakit diabetes. Inovasi ini memungkinkanperangkat perawatan kesehatan terhubung dengan jaringan internet, sehingga data pasien dapatdiperbaharui dan diakses secara real-time. Selain mempermudah akses, penggunaan IoT juga akanmemberikan nilai tambah pada efisiensi biaya pelayanan kesehatan. Penelitian ini bertujuan untukmerancang software sistem monitoring gula darah berbasis web yang terintegrasi dengan IoT,sehingga pasien dapat melakukan pemeriksaan, konsultasi dengan dokter dan melihat data rekammedis dari jarak jauh. Data hasil pemeriksaan akan disimpan didalam cloud dan ditampilkan secaraonline. Penelitian ini menggunakan Node MCU ESP8266 sebagai mikrokontroller yang telahdilengkapi dengan modul WiFi, Thingspeak sebagai cloud, aplikasi online dengan “Diamons” sebagaidashboard yang mampu menampilkan presentasi data grafis, dibangun dengan bahasa HypertextPreprocessor (PHP) sebagai bahasa pemogramannya. Penelitian ini akan melibatkan pihak medisdalam pengambilan keputusan. Umpan balik yang diberikan kepada pasien berupa anjuran sepertiresep obat, pola makan, dan kegiatan fisik yang harus dilakukan oleh pasien.

scholarly journals Circulation and Molecular Epidemiology of Enteroviruses in Paralyzed, Immunodeficient and Healthy Individuals in Tunisia, a Country with a Polio-Free Status for Decades

Viruses ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 380
Author(s):  
Anissa Chouikha ◽  
Dorra Rezig ◽  
Nadia Driss ◽  
Ichrak Abdelkhalek ◽  
Ahlem Ben Yahia ◽  
...  
Keyword(s):  
Detection Rate ◽  
Warning System ◽  
World Health ◽  
Clinical Samples ◽  
Nucleotide Sequencing ◽  
Comprehensive Picture ◽  
Set Up ◽  
Health Organization ◽  
Type 3

This report is an overview of enterovirus (EV) detection in Tunisian polio-suspected paralytic cases (acute flaccid paralysis (AFP) cases), healthy contacts and patients with primary immunodeficiencies (PID) during an 11-year period. A total of 2735 clinical samples were analyzed for EV isolation and type identification, according to the recommended protocols of the World Health Organization. Three poliovirus (PV) serotypes and 28 different nonpolio enteroviruses (NPEVs) were detected. The NPEV detection rate was 4.3%, 2.8% and 12.4% in AFP cases, healthy contacts and PID patients, respectively. The predominant species was EV-B, and the circulation of viruses from species EV-A was noted since 2011. All PVs detected were of Sabin origin. The PV detection rate was higher in PID patients compared to AFP cases and contacts (6.8%, 1.5% and 1.3% respectively). PV2 was not detected since 2015. Using nucleotide sequencing of the entire VP1 region, 61 strains were characterized as Sabin-like. Among them, six strains of types 1 and 3 PV were identified as pre-vaccine-derived polioviruses (VDPVs). Five type 2 PV, four strains belonging to type 1 PV and two strains belonging to type 3 PV, were classified as iVDPVs. The data presented provide a comprehensive picture of EVs circulating in Tunisia over an 11-year period, reveal changes in their epidemiology as compared to previous studies and highlight the need to set up a warning system to avoid unnoticed PVs.

Engaging older people in healthy and active lifestyles: a systematic review

2015 ◽  
Vol 36 (10) ◽  
pp. 2036-2060 ◽  
Author(s):  
JULIA MENICHETTI ◽  
PIETRO CIPRESSO ◽  
DARIO BUSSOLIN ◽  
GUENDALINA GRAFFIGNA
Keyword(s):  
Systematic Review ◽  
Health Promotion ◽  
World Health ◽  
Active Ageing ◽  
Individual Level ◽  
Active Lifestyles ◽  
Health Organization ◽  
The Individual ◽  
Health Promotion Interventions ◽  
Older Citizens

ABSTRACTIn 2002, the World Health Organization emphasised the concept of active ageing to manage and increase the last third of life. Although many efforts have been made to optimise treatment management, less attention has been paid to health promotion initiatives. To date, few shared guidelines exist that promote an active life in healthy older targets. To fill this gap, we conducted a systematic review to map health promotion interventions that targeted an active and healthy ageing among older citizens. Articles containing the key term active ageing and seven synonyms were searched for in the electronic databases. Because we were interested in actions aimed to promote healthier lifestyles, we connected the string with the term health. A total of 3,918 titles were retrieved and 20 articles were extracted. Twelve of the 20 studies used group interventions, five interventions targeted the individual level and three interventions targeted the community level. Interventions differed for the health focus of the programmes, which ranged from physical activity interventions to social participation or cognitive functioning. Most of the studies aimed to act on psychological components. The review suggests that different interventions promoted for active ageing are effective in improving specific healthy and active lifestyles; however, no studies were concerned directly with a holistic process of citizen health engagement to improve long-term outcomes.

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