Target Testing and Specificity of Nucleic Acid Based Diagnostics for COVID-19

Objective: The seek of this study is to provide an indication on the features of diagnostic testing of SARS-CoV-2 by RT-PCR, including parameters of sensitivity, specificity, positive and negative likelihood ratios. Background: Coronavirus Disease is the fifth international emergency after 1918 Spanish flu pandemic, triggered by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2). On 30 January, the WHO acknowledged COVID-19 to be a global health disaster of international importance and a pandemic on 11 March 2020. In vitro analyses of the data shows that for SARS-CoV-2 the RT-PCR test is highly specific, as it is not counter react with nucleic acid of other viruses. Methods: Oropharyngeal and nasopharyngeal swabs were collected into a 3 ml viral transport media (VTM) and transported to Laboratory. Extraction of the viral RNA was done by Qiasymphony DSP Virus/ Pathogen mini kit (Qiagen GmbH, Germany). For amplification process of RT-PCR qualitative detection of SARS-CoV-2 RNA utilizing with SYSTAAQ 2019-Novel Coronavirus (COVID-19) Real time PCR kit using a BIORAD-CFX 96. Results: Out of 15,049, 3195 samples were positive for covid-19 qPCR. Ratio of the Males patients were greater than females. 63.7% males and 36.3% females were effected with Covid-19. Symptom wise analysis shows 62% patient were asymptomatic, 22.7% mild, 1.7% moderate, 12.7% stable, 0.6% severe and 0.2% were critical. Our analysis reveals age group 1 (4.9%), group 2 (55.5%), group 3 (27.5%), and group 4 (12.1%) were effected with SARS-nCoV-2. Our result shows 3.0 % patients were deceased and 97% were recovered. Conclusion: Our findings contribute to the evolving understanding of the sophisticated interaction between this emerging SARS-CoV-2 virus and nucleic acid based target testing of COVID-19.

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Cases Report of Cured Novel Coronavirus-infected Pneumonia Patients with Viral Nucleic Acid Test Positive in Fecal Specimens

10.21203/rs.3.rs-20799/v1 ◽

2020 ◽

Author(s):

Mei Han ◽

Jingbo Zou ◽

Wenguang Tian ◽

Xiaoyu Wei ◽

Yang Zhou ◽

...

Keyword(s):

Nucleic Acid ◽

Molecular Diagnostic ◽

Clinical Practices ◽

Rt Pcr ◽

Viral Nucleic Acid ◽

Nucleic Acid Test ◽

Assessment Standard ◽

Novel Coronavirus ◽

Acid Test ◽

Respiratory Specimens

Abstract Background: The outbreak of the novel coronavirus in China (COVID-19) represents a significant and urgent threat to global health. We report here five cases of COVID-19 infection patients in our clinical practices who are medically stable and presumed to successfully “cleared” the virus after antiviral treatments. Case presentation: The clinical evaluation depends on the viral nucleic acid test in respiratory specimens by real-time PCR reverse transcription (RT-PCR) assays according to the authorized guidance. We found that the stool samples of these cured patients remain positive in RT-PCR assay while the virus is undetectable in respiratory specimens. RT-PCR molecular diagnostic assay was designed to specifically detect the presence of viral RNA. Thus, the positive result in the fecal specimens implies the existence of viable virions with the patients. Conclusions: This highlights the importance to look closely at the assessment standard of medical treatment, as well as the need for reevaluation of the criteria for the initial screening, prevention, and care of patients with this emerging infection.

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Has the Pandemic Triggered a ‘Paperdemic’? Towards an Assessment of Diagnostic Indicators for COVID-19

10.31219/osf.io/cyp8d ◽

2021 ◽

Author(s):

AISDL

Keyword(s):

Predictive Value ◽

Diagnostic Testing ◽

Supporting Evidence ◽

The Novel ◽

Scientific Publications ◽

Widespread Belief ◽

Novel Method ◽

Consistency Problem ◽

Novel Coronavirus ◽

Sensitivity Specificity

This paper is a preliminary step towards the assessment of an alarming widespread belief that victims of the novel coronavirus SARS-CoV-2 include the quality and accuracy of scientific publications about it. Our initial results suggest that this belief cannot be readily ignored, denied, dismissed or refuted, since some genuine supporting evidence can be forwarded for it. This evidence includes an obvious increase in retractions of papers published about the COVID-19 pandemic plus an extra-ordinary phenomenon of inconsistency that we report herein. In fact, we provide a novel method for validating any purported set of the four most prominent indicators of diagnostic testing (Sensitivity, Specificity, Positive Predictive Value, and Negative Predictive Value), by observing that these indicators constitute three rather than four independent quantities. This observation has virtually been unheard of in the open medical literature, and hence researchers have not taken it into consideration. We define two functions, which serve as consistency criteria, since each of them checks consistency for any set of four numerical values (naturally belonging to the interval [0.0,1.0]) claimed to be the four basic diagnostic indicators. Most of the data we came across in various international journals met our criteria for consistency, but in a few cases, there were obvious unexplained blunders. We explored the same consistency problem for some diagnostic data published in 2020 concerning the ongoing COVID-19 pandemic and observed that the afore-mentioned unexplained blunders tended to be on the rise. A systematic extensive statistical assessment of this resumed tendency is warranted.

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

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.015434 ◽

2020 ◽

Vol 295 (46) ◽

pp. 15438-15453 ◽

Cited By ~ 1

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.

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Detection and Quantification of the Red Tide Dinoflagellate Karenia brevis by Real-Time Nucleic Acid Sequence-Based Amplification

Applied and Environmental Microbiology ◽

10.1128/aem.70.8.4727-4732.2004 ◽

2004 ◽

Vol 70 (8) ◽

pp. 4727-4732 ◽

Cited By ~ 50

Author(s):

Erica T. Casper ◽

John H. Paul ◽

Matthew C. Smith ◽

Michael Gray

Keyword(s):

Nucleic Acid ◽

Real Time ◽

Wildlife Conservation ◽

Red Tide ◽

Diagnostic Testing ◽

Karenia Brevis ◽

Nucleic Acid Sequence ◽

Cell Counts ◽

Wide Range

ABSTRACT Nucleic acid sequence-based amplification (NASBA) is an isothermal method of RNA amplification that has been previously used in clinical diagnostic testing. A real-time NASBA assay has been developed for the detection of rbcL mRNA from the red tide dinoflagellate Karenia brevis. This assay is sensitive to one K. brevis cell and 1.0 fg of in vitro transcript, with occasional detection of lower concentrations of transcript. The assay did not detect rbcL mRNA from a wide range of nontarget organisms and environmental clones, while 10 strains (all tested) of K. brevis were detected. By the use of standard curves based on time to positivity, concentrations of K. brevis in environmental samples were predicted by NASBA and classified into different levels of blooms per the Florida Fish and Wildlife Conservation Commission (FWC) system. NASBA classification matched FWC classification (based on cell counts) 72% of the time. Those samples that did not match were off by only one class. NASBA is sensitive, rapid, and effective and may be used as an additional or alternative method to detect and quantify K. brevis in the marine environment.

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Validation of a Methodology for the Detection of Severe Acute Respiratory Syndrome Coronavirus 2 in Saliva by Real-Time Reverse Transcriptase-PCR

Frontiers in Public Health ◽

10.3389/fpubh.2021.743300 ◽

2021 ◽

Vol 9 ◽

Author(s):

Daniel F. Escobar ◽

Pablo Díaz ◽

Diego Díaz-Dinamarca ◽

Rodrigo Puentes ◽

Pedro Alarcón ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Reverse Transcriptase ◽

Nasopharyngeal Swab ◽

Rt Pcr ◽

Health Authorities ◽

Specific Alternative ◽

Pcr Method ◽

Sensitivity Specificity ◽

Study Participants

In January 2021, the Chilean city of Concepción experienced a second wave of coronavirus 2019 (COVID-19) while in early April 2021, the entire country faced the same situation. This outbreak generated the need to modify and validate a method for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in saliva, thereby expanding the capacity and versatility of testing for COVID-19. This study was conducted in February 2021 in the Chilean city of Concepción during which time, the town was under total quarantine. The study participants were mostly symptomatic (87.4%), not hospitalized, and attended care centers because of their health status rather than being asked by the researchers. People coming to the health center in Concepción to be tested for COVID-19 (via reverse transcriptase polymerase chain reaction [RT-PCR]) from a specimen of nasopharyngeal swab (NPS) were then invited to participate in this study. A total of 131 participants agreed to sign an informed consent and to provide saliva and NPS specimens to validate a method in terms of sensitivity, specificity, and statistical analysis of the cycle threshold (Ct) values from the RT-PCR. Calculations pertaining to the 127 participants who were ultimately included in the analysis showed sensitivity and specificity at 94.34% (95% CI: 84.34–98.82%) and 98.65% (95% CI: 92.70–99.97%), respectively. The saliva specimen showed a performance comparable to NPS as demonstrated by the diagnostic parameters. This RT-PCR method from the saliva specimen is a highly sensitive and specific alternative compared to the reference methodology, which uses the NPS specimen. This modified and validated method is intended for use in the in vitro diagnosis of SARS-CoV-2, which provides health authorities in Chile and local laboratories with a real testing alternative to RT-PCR from NPS.

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

Engineering Proceedings ◽

10.3390/i3s2021dresden-10076 ◽

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.

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Has the Pandemic Triggered a ‘Paperdemic’? Towards an Assessment of Diagnostic Indicators for COVID-19

International Journal of Pathogen Research ◽

10.9734/ijpr/2021/v6i230158 ◽

2021 ◽

pp. 28-49

Author(s):

Ali Muhammad Ali Rushdi ◽

Hamzah Abdul Majid Serag

Keyword(s):

Predictive Value ◽

Diagnostic Testing ◽

Supporting Evidence ◽

The Novel ◽

Scientific Publications ◽

Widespread Belief ◽

Novel Method ◽

Consistency Problem ◽

Novel Coronavirus ◽

Sensitivity Specificity

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Evaluation of a novel, rapid antigen detection test for the diagnosis of SARS-CoV-2

PLoS ONE ◽

10.1371/journal.pone.0259527 ◽

2021 ◽

Vol 16 (11) ◽

pp. e0259527

Author(s):

Rainer Thell ◽

Verena Kallab ◽

Wolfgang Weinhappel ◽

Wolfgang Mueckstein ◽

Lukas Heschl ◽

...

Keyword(s):

Emergency Departments ◽

Point Of Care ◽

Gain Control ◽

Rapid Test ◽

High Viral Load ◽

Rt Pcr ◽

Likelihood Ratios ◽

Predictive Values ◽

Rapid Antigen Detection Test ◽

Sensitivity Specificity

Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease 2019 (COVID-19) is currently finally determined in laboratory settings by real-time reverse-transcription polymerase-chain-reaction (rt-PCR). However, simple testing with immediately available results are crucial to gain control over COVID-19. The aim was to evaluate such a point-of-care antigen rapid test (AG-rt) device in its performance compared to laboratory-based rt-PCR testing in COVID-19 suspected, symptomatic patients. Methods For this prospective study, two specimens each of 541 symptomatic female (54.7%) and male (45.3%) patients aged between 18 and 95 years tested at five emergency departments (ED, n = 296) and four primary healthcare centres (PHC, n = 245), were compared, using AG-rt (positive/negative/invalid) and rt-PCR (positive/negative and cycle threshold, Ct) to diagnose SARS-CoV-2. Diagnostic accuracy, sensitivity, specificity, positive predictive values (PPV), negative predictive value (NPV), and likelihood ratios (LR+/-) of the AG-rt were assessed. Results Differences between ED and PHC were detected regarding gender, age, symptoms, disease prevalence, and diagnostic performance. Overall, 174 (32.2%) were tested positive on AG-rt and 213 (39.4%) on rt-PCR. AG correctly classified 91.7% of all rt-PCR positive cases with a sensitivity of 80.3%, specificity of 99.1%, PPV of 98.3, NPV of 88.6%, LR(+) of 87.8, and LR(-) of 0.20. The highest sensitivities and specificities of AG-rt were detected in PHC (sensitivity: 84.4%, specificity: 100.0%), when using Ct of 30 as cut-off (sensitivity: 92.5%, specificity: 97.8%), and when symptom onset was within the first three days (sensitivity: 82.9%, specificity: 99.6%). Conclusions The highest sensitivity was detected with a high viral load. Our findings suggest that AG-rt are comparable to rt-PCR to diagnose SARS-CoV-2 in COVID-19 suspected symptomatic patients presenting both at emergency departments and primary health care centres.

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Simultaneous Detection of 2019 Novel Coronavirus and Influenza Virus by Double Fluorescent RT-PCR

10.21203/rs.3.rs-18989/v1 ◽

2020 ◽

Author(s):

shasha zhang

Keyword(s):

Influenza Virus ◽

Nucleic Acid ◽

Simultaneous Detection ◽

Reaction System ◽

Type A ◽

Nucleic Acid Detection ◽

Nasopharyngeal Swab ◽

Rt Pcr ◽

Ct Value ◽

Novel Coronavirus

Abstract this paper introduces a method for simultaneous detection of 2019 novel coronavirus(2019-nCoV) and influenza virus by dual fluorescent RT-PCR, providing some references for the current clinical first-line practice against the epidemic. More than 1500 samples of nasopharyngeal swabs, sputum and anal swabs were collected. Nucleic acid detection kits from two manufacturers of novel coronavirus and type A/B influenza virus were selected for the detection. Carboxyfluorescein (FAM) and green fluorescent protein (VIC) labeled probes were used to achieve simultaneous detection of the four gene targets using a double fluorescent RT-PCR reaction system. According to the analysis for the results of nucleic acid detection of existing samples, there is no cross infection between 2019 novel coronavirus and type A/B influenza virus. The Ct value of novel coronavirus nucleic acid in anal swab>Ct value of sputum > Ct value of nasopharyngeal swab in the same patient. Conclusion: A method for rapid and simultaneous detection of novel coronavirus and influenza virus by dual fluorescent RT-PCR was established to improve the detection efficiency and reduce the cost, which could be used for rapid and emergent detection of 2019 novel coronavirus and type A/B influenza virus.

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496. Higher SARS-CoV-2 RT-PCR Cycle Threshold (Ct) Values Associated with Longer Symptom Duration Among Patients Hospitalized with COVID-19

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa439.689 ◽

2020 ◽

Vol 7 (Supplement_1) ◽

pp. S314-S314

Author(s):

Lillian B Brown ◽

Lisa Gail Winston ◽

Barbara Haller ◽

Phong Pham ◽

Beatrice Marcelo ◽

...

Keyword(s):

Viral Load ◽

San Francisco ◽

Symptom Onset ◽

Diagnostic Testing ◽

Symptom Duration ◽

Rt Pcr ◽

Duration Of Symptoms ◽

Cycle Threshold ◽

Asymptomatic Patients

Abstract Background Most diagnostic tests for SARS-CoV-2, the causative agent of COVID-19, are RT-PCR based. This method is sensitive but cannot distinguish replicating from non-replicating virus. RT-PCR cycle threshold (Ct) values are inversely correlated with viral load, and higher Ct values have been correlated with lower in vitro viral infectivity. However, relatively few data exist on the association between Ct values and patients’ duration of symptoms remains unclear. We thus evaluated Ct values and symptom duration in a cohort of patients hospitalized with COVID-19. Methods We assessed all patients admitted to San Francisco General Hospital between April 1 and May 18, 2020 with confirmed COVID-19 infection based on RT-PCR testing (Abbott m2000 platform). We included patients having diagnostic testing for suspected COVID-19 and patients having asymptomatic testing per hospital policy. For symptomatic patients, date of symptom onset was abstracted from hospital records, and time from symptom onset to test date was calculated. RT-PCR Ct values were manually extracted. Median Ct and IQR were calculated for patients with < 10 days of symptoms, ≥10 days of symptoms, and asymptomatic disease. Between-group comparisons were performed using the Kruskal-Wallis test. Results Among 61 patients with positive RT-PCR tests, 40 patients reported < 10 days of symptoms at the time of testing, 15 reported ≥10 days of symptoms, and 6 were asymptomatic. The median Ct value was 14.2 cycles (IQR, 10.2, 18.3) among patients reporting < 10 days of symptoms, 19.7 cycles (IQR, 15.3, 23.9) among patients reporting ≥10 days of symptoms, and 26.3 (IQR, 25.0, 29.1) among asymptomatic patients. Ct values were significantly lower among patients with < 10 days of symptoms compared to patients with >=10 days of symptoms (p=0.01) and when compared to asymptomatic patients (p=0.0002) [Figure]. Cycle threshold (Ct) by days of symptoms at time of testing Conclusion SARS-CoV-2 RT-PCR cycle threshold values were higher (indicating lower viral load) in patients with longer symptom duration and were highest in asymptomatic patients. These results add to emerging data suggesting that strategies for optimal isolation of patients in both community and hospital settings could be informed by a combination of symptom duration and RT-PCR Ct values. Disclosures All Authors: No reported disclosures

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