scholarly journals Evaluation of RNA extraction free method for detection of SARS-COV-2 in salivary samples for mass screening for COVID-19

2021 ◽  
Author(s):  
Sally A. Mahmoud ◽  
Esra Ibrahim ◽  
Subhashini Ganesan ◽  
Bhagyashree Thakre ◽  
Juliet G Teddy ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
Gold Standard ◽  
Rna Extraction ◽  
High Sensitivity ◽  
High Specificity ◽  
Cost Effective ◽  
Kappa Coefficient ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Good Percentage

AbstractIn this current COVID - 19 pandemic, there is a dire need for cost effective and less time-consuming alternatives for SARS-COV-2 testing. The RNA extraction free method for detecting SARS-COV-2 in saliva is a promising option, this study found that it has high sensitivity (85.34%), specificity (95.04%) and was comparable to the gold standard nasopharyngeal swab. The method showed good percentage of agreement (kappa coefficient) 0.797 between salivary and NPS samples. However, there are variations in the sensitivity and specificity based on the RT-PCR kit used. The Thermo Fischer-Applied biosystems showed high sensitivity, PPV and NPV but also showed higher percentage of invalid reports. Whereas the BGI kit showed high specificity, better agreement (kappa coefficient) between the results of saliva and NPS samples and higher correlation between the Ct values of saliva and NPS samples. Thus, the RNA extraction free method for salivary sample serves as an effective alternative for SARS-CoV 2-testing.

scholarly journals Evaluation of RNA Extraction-Free Method for Detection of SARS-CoV-2 in Salivary Samples for Mass Screening for COVID-19

2021 ◽  
Vol 2021 ◽  
pp. 1-6
Author(s):  
Sally A. Mahmoud ◽  
Subhashini Ganesan ◽  
Esra Ibrahim ◽  
Bhagyashree Thakre ◽  
Juliet G. Teddy ◽  
...  
Keyword(s):  
Predictive Value ◽  
Rna Extraction ◽  
Screening Method ◽  
High Sensitivity ◽  
High Specificity ◽  
Cost Effective ◽  
Kappa Coefficient ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Good Agreement

In this COVID-19 pandemic, there is a dire need for cost-effective and less time-consuming alternatives for SARS-CoV-2 testing. The RNA extraction-free method for detecting SARS-CoV-2 in saliva is a promising option. This study found that it has high sensitivity (85.34%), specificity (95.04%), and was comparable to the gold standard nasopharyngeal swab (NPS) sample tests. The method showed good agreement between salivary and NPS samples, with a kappa coefficient of 0.797. However, there are variations in the sensitivity and specificity based on the RT-PCR kit used. The Thermo Fisher Applied Biosystems showed high sensitivity, positive predictive value (PPV), and negative predictive value (NPV) but also showed a higher percentage of invalid reports. On the other hand, the BGI kit showed high specificity, better agreement (kappa coefficient) between the results of saliva and NPS samples, and higher correlation between the Ct values of saliva and NPS samples. Thus, the RNA extraction-free method for salivary sample serves as an effective alternative screening method for COVID-19.

scholarly journals Cost-Effective Pooling of DNA from Nasopharyngeal Swab Samples for Large-Scale Detection of Bacteria by Real-Time PCR

2014 ◽  
Vol 53 (3) ◽  
pp. 1002-1004 ◽  
Author(s):  
Sophie Edouard ◽  
Elsa Prudent ◽  
Philippe Gautret ◽  
Ziad A. Memish ◽  
Didier Raoult
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Large Scale ◽  
High Sensitivity ◽  
High Specificity ◽  
Cost Effective ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Scale Detection ◽  
The Cost

We investigated the potential of pooling DNA from nasopharyngeal specimens to reduce the cost of real-time PCR (RT-PCR) for bacterial detection. Lyophilization is required to reconcentrate DNA. This strategy yields a high specificity (86%) and a high sensitivity (96%). We estimate that compared to individual testing, 37% fewer RT-PCR tests are needed.

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 A scalable saliva-based, extraction-free rt-lamp protocol for sars-cov-2 diagnosis

2020 ◽  
Author(s):  
Paula Asprino ◽  
Fabiana Bettoni ◽  
Anamaria Camargo ◽  
Diego Coelho ◽  
Guilherme Coppini ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Cost Effective ◽  
Turnaround Time ◽  
Curve Analysis ◽  
Nasopharyngeal Swab ◽  
Screening Methods ◽  
Rt Pcr ◽  
Effective Screening ◽  
Point Curve ◽  
Average Turnaround Time

I.ABSTRACTScalable, cost-effective screening methods are an essential tool to control SARS-CoV-2 spread. We have developed a straight saliva-based, RNA extraction-free, RT-LAMP test that is comparable to current nasopharyngeal swab RT-PCR tests in both sensitivity and specificity. Using a 2-step readout of fluorescence and melting-point curve analysis, the test is scalable to more than 30,000 tests per day with average turnaround time of less than 3 hours. The test was validated using samples from 244 symptomatic patients, and showed sensitivity of 78.9% (vs. 85.5% for nasopharyngeal swabs RT-PCR) and specificity of 100% (vs. 100% for nasopharyngeal swabs RT-PCR). Our method is therefore accurate, robust, time and cost effective and therefore can be used for screening of SARS-CoV-2.

scholarly journals A new Syber Green real time PCR to detect SARS-CoV-2

2020 ◽  
Author(s):  
D.R. Marinowic ◽  
G. Zanirati ◽  
F.V.F. Rodrigues ◽  
M.V.C. Grahl ◽  
A.M. Alcará ◽  
...  
Keyword(s):  
Diagnostic Test ◽  
Large Scale ◽  
Low Cost ◽  
Phylogenetic Analyses ◽  
High Sensitivity ◽  
High Specificity ◽  
Cost Effective ◽  
Etiologic Agent ◽  
Rt Pcr ◽  
Human Respiratory Tract

Abstract Phylogenetic analyses demonstrated that etiologic agent of pandemic outbreak is a betacoronavirus named SARS-CoV-2. For public health interventions, a diagnostic test with high sensitivity and specificity is required. The gold standard protocol for diagnosis by WHO is the RT-PCR. To detect low viral load and large-scale screening a low-cost diagnostic test becomes necessary. Here we develop a cost-effective test capable of to detect the new coronavirues. We validated an auxiliary protocol for molecular diagnosis with RT-PCR SYBR Green methodology to successfully screen negative cases of SARS-CoV-2. Our results demonstrated that a set of primers with high specificity, and no homology with other viruses from Coronovideae family or human respiratory tract pathogenic viruses. Optimization of annealing temperature and polymerization time led to an high specificity in the PCR products. We have developed a more affordable and swift methodology for negative SARS-CoV-2 screening. This methodology can be applied on large scale populational to soften panic and economic burden through guidance for isolation strategies.

scholarly journals Combining machine learning and nanopore construction creates an artificial intelligence nanopore for coronavirus detection

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Masateru Taniguchi ◽  
Shohei Minami ◽  
Chikako Ono ◽  
Rina Hamajima ◽  
Ayumi Morimura ◽  
...  
Keyword(s):  
Artificial Intelligence ◽  
Machine Learning ◽  
High Speed ◽  
Simple Procedure ◽  
Rna Extraction ◽  
Disease Outbreaks ◽  
High Sensitivity ◽  
Cost Effective ◽  
Rt Pcr ◽  
Emerging Viruses

AbstractHigh-throughput, high-accuracy detection of emerging viruses allows for the control of disease outbreaks. Currently, reverse transcription-polymerase chain reaction (RT-PCR) is currently the most-widely used technology to diagnose the presence of SARS-CoV-2. However, RT-PCR requires the extraction of viral RNA from clinical specimens to obtain high sensitivity. Here, we report a method for detecting novel coronaviruses with high sensitivity by using nanopores together with artificial intelligence, a relatively simple procedure that does not require RNA extraction. Our final platform, which we call the artificially intelligent nanopore, consists of machine learning software on a server, a portable high-speed and high-precision current measuring instrument, and scalable, cost-effective semiconducting nanopore modules. We show that artificially intelligent nanopores are successful in accurately identifying four types of coronaviruses similar in size, HCoV-229E, SARS-CoV, MERS-CoV, and SARS-CoV-2. Detection of SARS-CoV-2 in saliva specimen is achieved with a sensitivity of 90% and specificity of 96% with a 5-minute measurement.

scholarly journals Mass molecular testing for COVID19 using NGS-based technology and a highly scalable workflow

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Fernanda de Mello Malta ◽  
Deyvid Amgarten ◽  
Felipe Camilo Val ◽  
Murilo Castro Cervato ◽  
Bruna Mascaro Cordeiro de Azevedo ◽  
...  
Keyword(s):  
Gold Standard ◽  
Rna Extraction ◽  
Cost Effective ◽  
Single Step ◽  
Molecular Testing ◽  
Rt Pcr ◽  
Major Bottleneck ◽  
Wet Lab ◽  
Coronavirus Infection ◽  
Performance Results

AbstractSince the first reported case of the new coronavirus infection in Wuhan, China, researchers and governments have witnessed an unseen rise in the number of cases. Thanks to the rapid work of Chinese scientists, the pathogen now called SARS-CoV-2 has been identified and its whole genome was deposited in public databases by early January 2020. The availability of the genome has allowed researchers to develop Reverse Transcription—Polymerase Chain Reaction (RT-PCR) assays, which are now the gold-standard for molecular diagnosis of the respiratory syndrome COVID19. Because of the rising number of cases and rapid spreading, the world has been facing a shortage of RT-PCR supplies, especially the ones involved in RNA extraction. This has been a major bottleneck to increase testing capacity in many countries that do not significantly manufacture these supplies, such as Brazil. Additionally, RT-qPCR scalability is highly dependent on equipment that usually performs testing of 96 samples at a time. In this work, we describe a cost-effective molecular NGS-based test for diagnosis of COVID19, which uses a single-step RNA extraction and presents high scalability and accuracy when compared to the gold-standard RT-qPCR. A single run of the NGS-based test using the Illumina NextSeq 550 mid-end sequencing equipment is able to multiplex 1,536 patient’s samples, providing individual semi-qualitative results (detected, not detected). Detected results are provided with fragments per million (FPM) values, which was demonstrated to correlate with RT-qPCR Cycle Threshold (CT) values. Besides, usage of the high-end Illumina Novaseq platform may yield diagnostic for up to 6144 samples in a single run. Performance results when compared with RT-qPCR show general accuracy of 96%, and 98% when only samples with CT values (gene N) lower than 30 are considered. We have also developed an online platform, termed VarsVID, to help test executors to easily scale testing numbers. Sample registering, wet-lab worksheets generation, sample sheet for sequencing and results’ display are all features provided by VarsVID. Altogether, these results will contribute to control COVID19 pandemics.

scholarly journals A new SYBR Green real-time PCR to detect SARS-CoV-2

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
D. R. Marinowic ◽  
G. Zanirati ◽  
F. V. F. Rodrigues ◽  
M. V. C. Grahl ◽  
A. M. Alcará ◽  
...  
Keyword(s):  
Diagnostic Test ◽  
Large Scale ◽  
Low Cost ◽  
High Sensitivity ◽  
High Specificity ◽  
Cost Effective ◽  
Etiologic Agent ◽  
Sybr Green ◽  
Rt Pcr ◽  
Human Respiratory Tract

AbstractPhylogenetic analysis has demonstrated that the etiologic agent of the 2020 pandemic outbreak is a betacoronavirus named SARS-CoV-2. For public health interventions, a diagnostic test with high sensitivity and specificity is required. The gold standard protocol for diagnosis by the Word Health Organization (WHO) is RT-PCR. To detect low viral loads and perform large-scale screening, a low-cost diagnostic test is necessary. Here, we developed a cost-effective test capable of detecting SARS-CoV-2. We validated an auxiliary protocol for molecular diagnosis with the SYBR Green RT-PCR methodology to successfully screen negative cases of SARS-CoV-2. Our results revealed a set of primers with high specificity and no homology with other viruses from the Coronovideae family or human respiratory tract pathogenic viruses, presenting with complementarity only for rhinoviruses/enteroviruses and Legionella spp. Optimization of the annealing temperature and polymerization time led to a high specificity in the PCR products. We have developed a more affordable and swift methodology for negative SARS-CoV-2 screening. This methodology can be applied on a large scale to soften panic and economic burden through guidance for isolation strategies.

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 Mass molecular testing for COVID19 using NGS-based technology and a highly scalable workflow

2020 ◽  
Author(s):  
Fernanda de Mello Malta ◽  
Deyvid Emanuel Amgarten ◽  
Felipe Camilo Val ◽  
Rubia Anita Ferraz Santana ◽  
Murilo Castro Cervato ◽  
...  
Keyword(s):  
Gold Standard ◽  
Rna Extraction ◽  
Cost Effective ◽  
Single Step ◽  
Molecular Testing ◽  
Rt Pcr ◽  
Major Bottleneck ◽  
Wet Lab ◽  
Coronavirus Infection ◽  
Performance Results

Since first reported case of the new coronavirus infection in Wuhan, China, researchers and governments have witnessed an unseen rise in the number of cases. Thanks to the rapid work of Chinese scientists, the pathogen now called SARS-CoV-2 has been identified and its whole genome has been deposited in public databases by early January 2020. The availability of the genome has allowed researchers to develop Reverse Transcription - Polymerase Chain Reaction (RT-PCR) assays, which are now the gold-standard for molecular diagnosis of the respiratory syndrome COVID19. Because of the rising number of cases and rapid spreading, the world has been facing a shortage of RT-PCR supplies, especially the ones involved in RNA extraction. This has been a major bottleneck to increase testing capacity in many countries that do not significantly manufacture these supplies (Brazil included). Additionally, RT-PCR scalability is highly dependent on equipment that usually perform testing of 96 samples at a time. In this work, we describe a cost-effective molecular NGS-based test for diagnosis of COVID19, which uses a single-step RNA extraction and presents high scalability and accuracy when compared to the gold-standard RT-PCR. A single run of the NGS-based test using the Illumina NextSeq 550 mid-end sequencing equipment is able to multiplex 1,536 patient samples, providing individual semi-qualitative results (detected, not detected). Detected results are provided with fragments per million (FPM) values, which was demonstrated to correlate with RT-PCR Cycle Threshold (CT) values. Besides, usage of the high-end Illumina Novaseq platform may yield diagnostic for up to 6,144 samples in a single run. Performance results when compared with RT-PCR show general accuracy of 96% (or 98% when only samples with CT values for gene N lower than 30 are considered). We have also developed an online platform, called VarsVID, a Varstation feature, to help test executors to easily scale testing numbers. Sample registering, wet-lab worksheets, sample sheet for sequencing and results display are all features provided by VarsVID on Varstation. Altogether, these results will contribute to control COVID19 pandemics.

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