scholarly journals LATE-PCR for LoC Molecular Diagnostics Devices and Its Application to the Sensitive Detection of SARS-CoV-2

2021 ◽  
Vol 6 (1) ◽  
pp. 43
Author(s):  
Dimitrios Karadimas ◽  
George Tsekenis
Keyword(s):  
Point Of Care ◽  
Low Cost ◽  
Diagnostic Testing ◽  
Heat Denaturation ◽  
Nasopharyngeal Swab ◽  
The Novel ◽  
Rt Pcr ◽  
Qualitative Detection ◽  
Novel Coronavirus ◽  
Real Patients

The emergence of the novel coronavirus, SARS-CoV-2, has highlighted the need for rapid, accurate, and point-of-care diagnostic testing. Lab-on-a-Chip (LoC) devices offer the possibility to run such tests at a low cost, while at the same time permitting the multiplexed detection of several viruses when coupled with microarray detection of the amplified products. Herein, we report the development of a protocol for the qualitative detection of SARS-CoV-2, through the design of appropriate primers that target the evolutionary conserved regions of the virus. The proposed protocol relies on an improved version of asymmetric RT-PCR, the linear-after-the-exponential (LATE)-PCR that uses primers that are deliberately designed for use at unequal concentrations. As a result, LATE-PCR exhibits similar efficiency to symmetric PCR, while promoting accumulation of single-stranded products that can subsequently hybridize to a single-strand DNA probe-spotted microarray. The performance of the developed LATE-PCR protocol was compared to that of symmetric RT-PCR, and validated with the use of artificial viral RNA and nasopharyngeal swab samples from real patients. Furthermore, and in order to illustrate its potential for integration into a biosensor platform, the amplicons were allowed to hybridize with probes that were covalently immobilized onto commercially available functionalized glass, without the need for heat denaturation.

scholarly journals A blueprint for academic laboratories to produce SARS-CoV-2 quantitative RT-PCR test kits

2020 ◽  
Vol 295 (46) ◽  
pp. 15438-15453 ◽  
Author(s):  
Samantha J. Mascuch ◽  
Sara Fakhretaha-Aval ◽  
Jessica C. Bowman ◽  
Minh Thu H. Ma ◽  
Gwendell Thomas ◽  
...  
Keyword(s):  
Support Group ◽  
Diagnostic Testing ◽  
The Novel ◽  
Rt Pcr ◽  
Research Teams ◽  
Environmental Testing ◽  
Resource Limited ◽  
Test Kit ◽  
Institute Of Technology ◽  
Novel Coronavirus

Widespread testing for the presence of the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in individuals remains vital for controlling the COVID-19 pandemic prior to the advent of an effective treatment. Challenges in testing can be traced to an initial shortage of supplies, expertise, and/or instrumentation necessary to detect the virus by quantitative RT-PCR (RT-qPCR), the most robust, sensitive, and specific assay currently available. Here we show that academic biochemistry and molecular biology laboratories equipped with appropriate expertise and infrastructure can replicate commercially available SARS-CoV-2 RT-qPCR test kits and backfill pipeline shortages. The Georgia Tech COVID-19 Test Kit Support Group, composed of faculty, staff, and trainees across the biotechnology quad at Georgia Institute of Technology, synthesized multiplexed primers and probes and formulated a master mix composed of enzymes and proteins produced in-house. Our in-house kit compares favorably with a commercial product used for diagnostic testing. We also developed an environmental testing protocol to readily monitor surfaces for the presence of SARS-CoV-2. Our blueprint should be readily reproducible by research teams at other institutions, and our protocols may be modified and adapted to enable SARS-CoV-2 detection in more resource-limited settings.

scholarly journals Recurrence of COVID-19 related symptoms and viral detection in patient discharged after complete recovery and test negativization

2021 ◽  
Author(s):  
Chiara Vassallo ◽  
Francesca Pupo ◽  
Luca Marri ◽  
Chiara Schiavi ◽  
Francesca Giusti ◽  
...  
Keyword(s):  
Complete Recovery ◽  
Viral Reactivation ◽  
Nasopharyngeal Swab ◽  
Double Negative ◽  
The Novel ◽  
Lung Involvement ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Virus Escape ◽  
Novel Coronavirus

Since the novel coronavirus disease 2019 (COVID-19) has declared pandemic, the possibility of recurrence of the disease after recovery has become a debated issue. We report a case of an 84-yearsold male patient who was admitted to our hospital for dyspnea and fever. Lab and clinical workout showed that he had COVID-19. After a full recovery of symptoms and a double negative nasopharyngeal swab of SARS-CoV-2 by RT-PCR assay, he was dismissed from the hospital. One month later, he developed again dyspnea and fever with lung involvement. Surprisingly, nasopharyngeal swab of SARS-CoV-2 was positive. Since he denied contacts with confirmed or suspected cases of COVID-19, he probably experienced a reactivation of a persistent infection. The failed eradication of the virus could depend on both virus’ escape mechanisms and dysfunctional immune response. Further studies are needed to confirm the hypothesis of viral reactivation and to identify signs of an incomplete clearance.

scholarly journals Nanobiosensors as new diagnostic tools for SARS, MERS and COVID-19: from past to perspectives

2020 ◽  
Vol 187 (12) ◽  
Author(s):  
Riccarda Antiochia
Keyword(s):  
State Of The Art ◽  
Point Of Care ◽  
Respiratory Illness ◽  
Diagnostic Tools ◽  
The Novel ◽  
Rt Pcr ◽  
Critical Comparison ◽  
Novel Coronavirus ◽  
And Control ◽  
Affinity Biosensors

AbstractThe severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS) and novel coronavirus 19 (COVID-19) epidemics represent the biggest global health threats in the last two decades. These infections manifest as bronchitis, pneumonia or severe, sometimes fatal, respiratory illness. The novel coronavirus seems to be associated with milder infections but it has spread globally more rapidly becoming a pandemic. This review summarises the state of the art of nanotechnology-based affinity biosensors for SARS, MERS and COVID-19 detection. The nanobiosensors are antibody- or DNA-based biosensors with electrochemical, optical or FET-based transduction. Various kinds of nanomaterials, such as metal nanoparticles, nanowires and graphene, have been merged to the affinity biosensors to enhance their analytical performances. The advantages of the use of the nanomaterials are highlighted, and the results compared with those obtained using non-nanostructured biosensors. A critical comparison with conventional methods, such as RT-PCR and ELISA, is also reported. It is hoped that this review will provide interesting information for the future development of new reliable nano-based platforms for point-of-care diagnostic devices for COVID-19 prevention and control.

scholarly journals Comparison of clinical severity and epidemiological spectrum between coronavirus disease 2019 and influenza in children

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Maria Pokorska-Śpiewak ◽  
Ewa Talarek ◽  
Jolanta Popielska ◽  
Karolina Nowicka ◽  
Agnieszka Ołdakowska ◽  
...  
Keyword(s):  
Clinical Presentation ◽  
Clinical Course ◽  
Household Contact ◽  
Clinical Severity ◽  
Nasopharyngeal Swab ◽  
Suspected Case ◽  
The Novel ◽  
Rt Pcr ◽  
Main Risk Factor ◽  
Novel Coronavirus

AbstractData on the novel coronavirus disease 2019 (COVID-19) in children are limited, and studies from Europe are scarce. We analyzed the clinical severity and epidemiologic aspects of COVID-19 in consecutive children aged 0–18 years, referred with a suspicion of COVID-19 between February 1, and April 15, 2020. RT-PCR on a nasopharyngeal swab was used to confirm COVID-19. 319 children met the criteria of a suspected case. COVID-19 was diagnosed in 15/319 (4.7%) patients (8 male; mean age 10.5 years). All of them had household contact with an infected relative. Five (33.3%) patients were asymptomatic. In 9/15 (60.0%) children, the course of the disease was mild, and in 1/15 (6.7%), it was moderate, with the following symptoms: fever (46.7%), cough (40%), diarrhea (20%), vomiting (13.3%), rhinitis (6.7%), and shortness of breath (6.7%). In the COVID-19-negative patients, other infections were confirmed, including influenza in 32/319 (10%). The clinical course of COVID-19 and influenza differed significantly based on the clinical presentation. In conclusion, the clinical course of COVID-19 in children is usually mild or asymptomatic. In children suspected of having COVID-19, other infections should not be overlooked. The main risk factor for COVID-19 in children is household contact with an infected relative.

scholarly journals Chest Radiograph (CXR) Manifestations of the Novel Coronavirus Disease 2019 (Covid-19) — A Mini-Review

2020 ◽  
Vol 16 ◽  
Author(s):  
Wai Yee Chan ◽  
Marlina Tanty Ramli Hamid ◽  
Nadia Fareeda Muhammad Gowdh ◽  
Kartini Rahmat ◽  
Nur Adura Yaakup ◽  
...  
Keyword(s):  
Disease Progression ◽  
Real Time ◽  
Low Cost ◽  
Tertiary Care ◽  
Severe Illness ◽  
Care Hospital ◽  
The Novel ◽  
Rt Pcr ◽  
Chest X Ray ◽  
Novel Coronavirus

Background: Coronavirus disease 2019 (COVID-19) is highly contagious and has claimed more than one million lives, besides causing hardship and disruptions. The Fleischner Society has recommended chest X-ray (CXR) in detecting cases with high risk for disease progression, for triaging suspected patients with moderate-to-severe illness, and to eliminate false negatives in areas with high pre-test probability or limited resources. Although CXR is less sensitive than real-time reverse transcription polymerase chain reaction (RT-PCR) in detecting mild COVID-19, it is nevertheless useful because of equipment portability, low cost and practicality in serial assessments of disease progression among hospitalized patients. Objective: This study aims to review the typical and relatively atypical CXR manifestations of COVID-19 pneumonia in a tertiary care hospital. Methods: The CXRs of 136 COVID-19 patients confirmed through real-time RT-PCR from March to May 2020 were reviewed. Literature search was performed using PubMed. Results: A total of 54 patients had abnormal CXR whilst the others were normal. Typical CXR findings included pulmonary consolidation or ground-glass opacities in a multifocal, bilateral peripheral or lower zone distribution, whereas atypical CXR features comprised cavitation and pleural effusion. Conclusion: Typical findings of COVID-19 infection in chest computed tomography studies can also be seen in CXR. The presence of atypical features is associated with worse disease outcome. Recognition of these features on CXR will improve accuracy and speed of diagnosing COVID-19 patients.

scholarly journals Direct diagnostic testing of SARS-CoV-2 without the need for prior RNA extraction

2020 ◽  
Author(s):  
Shan Wei ◽  
Esther Kohl ◽  
Alexandre Djandji ◽  
Stephanie Morgan ◽  
Susan Whittier ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Point Of Care ◽  
Low Cost ◽  
Diagnostic Testing ◽  
Rna Extraction ◽  
Nucleic Acid Amplification ◽  
Nasopharyngeal Swab ◽  
Viral Transport ◽  
Extraction Step ◽  
Highly Sensitive

AbstractThe COVID-19 pandemic has resulted in an urgent global need for rapid, point-of-care diagnostic testing. Existing methods for nucleic acid amplification testing (NAAT) require an RNA extraction step prior to amplification of the viral RNA. This step necessitates the use of a centralized laboratory or complex and costly proprietary cartridges and equipment, and thereby prevents low-cost, scalable, point-of-care testing. We report the development of a highly sensitive and robust, easy-to-implement, SARS-CoV-2 test that utilizes isothermal amplification and can be run directly on viral transport media following a nasopharyngeal swab without the need for prior RNA extraction. Our assay provides visual results in 30 min with 85% sensitivity, 100% specificity, and a limit of detection (LoD) of 2.5 copies/μl, and can be run using a simple heat block.

scholarly journals Extended Storage of SARS-CoV2 Nasopharyngeal Swabs Does Not Negatively Impact Results of Molecular-Based Testing

2020 ◽  
Author(s):  
Karin A Skalina ◽  
D. Yitzchak Goldstein ◽  
Jaffar Sulail ◽  
Eunkyu Hahm ◽  
Momka Narlieva ◽  
...  
Keyword(s):  
Nasopharyngeal Swab ◽  
Molecular Testing ◽  
The Novel ◽  
Clinical Tests ◽  
Ambient Conditions ◽  
Rt Pcr ◽  
Long Term Stability ◽  
Novel Coronavirus ◽  
World Environment

With the global outbreak of the novel coronavirus disease 2019, the demand for testing rapidly increased and quickly exceeded the testing capacities for many laboratories. Clinical tests which receive CE and FDA authorizations cannot always be tested thoroughly in a real-world environment. Here we demonstrate the long-term stability of nasopharyngeal swab specimens for SARS-CoV-2 molecular testing across three assays recently approved by the U.S. FDA under Emergency Use Authorization. This study demonstrates that nasopharyngeal swab specimens can be stored under refrigeration or even ambient conditions for 21 days without clinically impacting the results of the real-time RT-PCR testing.

scholarly journals SARS-CoV-2 Is Not Detected in the Cerebrospinal Fluid of Encephalopathic COVID-19 Patients

2020 ◽  
Vol 11 ◽  
Author(s):  
Dimitris G. Placantonakis ◽  
Maria Aguero-Rosenfeld ◽  
Abdallah Flaifel ◽  
John Colavito ◽  
Kenneth Inglima ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Viral Entry ◽  
Cell Types ◽  
Host Cells ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Neurologic Sequelae ◽  
Show Evidence ◽  
Novel Coronavirus ◽  
Transmembrane Serine Protease

Neurologic manifestations of the novel coronavirus SARS-CoV-2 infection have received wide attention, but the mechanisms remain uncertain. Here, we describe computational data from public domain RNA-seq datasets and cerebrospinal fluid data from adult patients with severe COVID-19 pneumonia that suggest that SARS-CoV-2 infection of the central nervous system is unlikely. We found that the mRNAs encoding the ACE2 receptor and the TMPRSS2 transmembrane serine protease, both of which are required for viral entry into host cells, are minimally expressed in the major cell types of the brain. In addition, CSF samples from 13 adult encephalopathic COVID-19 patients diagnosed with the viral infection via nasopharyngeal swab RT-PCR did not show evidence for the virus. This particular finding is robust for two reasons. First, the RT-PCR diagnostic was validated for CSF studies using stringent criteria; and second, 61% of these patients had CSF testing within 1 week of a positive nasopharyngeal diagnostic test. We propose that neurologic sequelae of COVID-19 are not due to SARS-CoV-2 meningoencephalitis and that other etiologies are more likely mechanisms.

scholarly journals 458. Molecular SARS-CoV-2 Testing During the COVID-19 Outbreak: Experiences of a Hospital in Southeast Michigan, USA

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S296-S297
Author(s):  
Trini A Mathew ◽  
Jonathan Hopkins ◽  
Diane Kamerer ◽  
Shagufta N Ali ◽  
Daniel Ortiz ◽  
...  
Keyword(s):  
Clinical Features ◽  
Diagnostic Testing ◽  
Beaumont Hospital ◽  
High Capacity ◽  
Turnaround Time ◽  
Molecular Diagnostic ◽  
The Novel ◽  
Repeat Testing ◽  
Asymptomatic Patients ◽  
Novel Coronavirus

Abstract Background The novel Coronavirus SARS CoV-2 (COVID-19) outbreak was complicated by the lack of diagnostic testing kits. In early March 2020, leadership at Beaumont Hospital, Royal Oak Michigan (Beaumont) identified the need to develop high capacity testing modalities with appropriate sensitivity and specificity and rapid turnaround time. We describe the molecular diagnostic testing experience since initial rollout on March 16, 2020 at Beaumont, and results of repeat testing during the peak of the COVID-19 pandemic in MI. Methods Beaumont is an 1100 bed hospital in Southeast MI. In March, testing was initially performed with the EUA Luminex NxTAG CoV Extended Panel until March 28, 2020 when testing was converted to the EUA Cepheid Xpert Xpress SARS-CoV-2 for quicker turnaround times. Each assay was validated with a combination of patient samples and contrived specimens. Results During the initial week of testing there was > 20 % specimen positivity. As the prevalence grew the positivity rate reached 68% by the end of March (Figure 1). Many state and hospital initiatives were implemented during the outbreak, including social distancing and screening of asymptomatic patients to increase case-finding and prevent transmission. We also adopted a process for clinical review of symptomatic patients who initially tested negative for SARS-CoV-2 by a group of infectious disease physicians (Figure 2). This process was expanded to include other trained clinicians who were redeployed from other departments in the hospital. Repeat testing was performed to allow consideration of discontinuation of isolation precautions. During the surge of community cases from March 16 to April 30, 2020, we identified patients with negative PCR tests who subsequently had repeat testing based on clinical evaluation, with 7.1% (39/551) returning positive for SARS- CoV2. Of the patients who expired due to COVID-19 during this period, 4.3% (9/206) initially tested negative before ultimately testing positive. Figure 1 BH RO testing Epicurve Figure 2: Screening tool for repeat COVID19 testing and precautions Conclusion Many state and hospital initiatives helped us flatten the curve for COVID-19. Our hospital testing experience indicate that repeat testing may be warranted for those patients with clinical features suggestive of COVID-19. We will further analyze these cases and clinical features that prompted repeat testing. Disclosures All Authors: No reported disclosures

scholarly journals Direct antivirals working against the novel coronavirus: azithromycin (DAWn-AZITHRO), a randomized, multicenter, open-label, adaptive, proof-of-concept clinical trial of new antivirals working against SARS-CoV-2—azithromycin trial

Trials ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Iwein Gyselinck ◽  
◽  
Laurens Liesenborghs ◽  
Ewout Landeloos ◽  
Ann Belmans ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Standard Of Care ◽  
Ordinal Scale ◽  
Cytokine Signaling ◽  
Nasopharyngeal Swab ◽  
The Novel ◽  
Proof Of Concept ◽  
Open Label ◽  
Novel Coronavirus

Abstract Background The rapid emergence and the high disease burden of the novel coronavirus SARS-CoV-2 have created a medical need for readily available drugs that can decrease viral replication or blunt the hyperinflammatory state leading to severe COVID-19 disease. Azithromycin is a macrolide antibiotic, known for its immunomodulatory properties. It has shown antiviral effect specifically against SARS-CoV-2 in vitro and acts on cytokine signaling pathways that have been implicated in COVID-19. Methods DAWn-AZITHRO is a randomized, open-label, phase 2 proof-of-concept, multicenter clinical trial, evaluating the safety and efficacy of azithromycin for treating hospitalized patients with COVID-19. It is part of a series of trials testing promising interventions for COVID-19, running in parallel and grouped under the name DAWn-studies. Patients hospitalized on dedicated COVID wards are eligible for study inclusion when they are symptomatic (i.e., clinical or radiological signs) and have been diagnosed with COVID-19 within the last 72 h through PCR (nasopharyngeal swab or bronchoalveolar lavage) or chest CT scan showing typical features of COVID-19 and without alternate diagnosis. Patients are block-randomized (9 patients) with a 2:1 allocation to receive azithromycin plus standard of care versus standard of care alone. Standard of care is mostly supportive, but may comprise hydroxychloroquine, up to the treating physician’s discretion and depending on local policy and national health regulations. The treatment group receives azithromycin qd 500 mg during the first 5 consecutive days after inclusion. The trial will include 284 patients and recruits from 15 centers across Belgium. The primary outcome is time from admission (day 0) to life discharge or to sustained clinical improvement, defined as an improvement of two points on the WHO 7-category ordinal scale sustained for at least 3 days. Discussion The trial investigates the urgent and still unmet global need for drugs that may impact the disease course of COVID-19. It will either provide support or else justify the discouragement of the current widespread, uncontrolled use of azithromycin in patients with COVID-19. The analogous design of other parallel trials of the DAWN consortium will amplify the chance of identifying successful treatment strategies and allow comparison of treatment effects within an identical clinical context. Trial registration EU Clinical trials register EudraCT Nb 2020-001614-38. Registered on 22 April 2020

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