scholarly journals SARSeq, a robust and highly multiplexed NGS assay for parallel detection of SARS-CoV2 and other respiratory infections

2020 ◽  
Author(s):  
Ramesh Yelagandula ◽  
Aleksandr Bykov ◽  
Alexander Vogt ◽  
Robert Heinen ◽  
Ezgi Özkan ◽  
...  
Keyword(s):  
Influenza A ◽  
Respiratory Infections ◽  
False Positive Rate ◽  
Cost Effective ◽  
Rt Pcr ◽  
Robust Performance ◽  
Saliva Analysis ◽  
Positive Rate ◽  
Double Blinded ◽  
Generation Sequencing

During a pandemic, mitigation as well as protection of system-critical or vulnerable institutions requires massively parallel, yet cost-effective testing to monitor the spread of agents such as the current SARS-CoV2 virus. Here we present SARSeq, saliva analysis by RNA sequencing, as an approach to monitor presence of SARS-CoV2 and other respiratory viruses performed on tens of thousands of samples in parallel. SARSeq is based on next generation sequencing of multiple amplicons generated in parallel in a multiplexed RT-PCR reaction. It relies on a two-dimensional unique dual indexing strategy using four indices in total, for unambiguous and scalable assignment of reads to individual samples. We calibrated this method using dilutions of synthetic RNA and virions to show sensitivity down to a few molecules, and applied it to hundreds of patient samples validating robust performance across various sample types. Double blinded benchmarking to gold-standard quantitative RT-PCR performed in a clinical setting and a human diagnostics laboratory showed robust performance up to a Ct of 36. The false positive rate, likely due to cross contamination during sample pipetting, was estimated at 0.04-0.1%. In addition to SARS-CoV2, SARSeq detects Influenza A and B viruses as well as human rhinovirus and can be easily expanded to include detection of other pathogens. In sum, SARSeq is an ideal platform for differential diagnostic of respiratory diseases at a scale, as is required during a pandemic.

scholarly journals Multiplexed detection of SARS-CoV-2 and other respiratory infections in high throughput by SARSeq

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ramesh Yelagandula ◽  
◽  
Aleksandr Bykov ◽  
Alexander Vogt ◽  
Robert Heinen ◽  
...  
Keyword(s):  
Influenza A ◽  
Respiratory Infections ◽  
High Sensitivity ◽  
Cost Effective ◽  
Massively Parallel ◽  
Rt Pcr ◽  
Viral Spread ◽  
Saliva Analysis ◽  
Double Blinded ◽  
Generation Sequencing

AbstractThe COVID-19 pandemic has demonstrated the need for massively-parallel, cost-effective tests monitoring viral spread. Here we present SARSeq, saliva analysis by RNA sequencing, a method to detect SARS-CoV-2 and other respiratory viruses on tens of thousands of samples in parallel. SARSeq relies on next generation sequencing of multiple amplicons generated in a multiplexed RT-PCR reaction. Two-dimensional, unique dual indexing, using four indices per sample, enables unambiguous and scalable assignment of reads to individual samples. We calibrate SARSeq on SARS-CoV-2 synthetic RNA, virions, and hundreds of human samples of various types. Robustness and sensitivity were virtually identical to quantitative RT-PCR. Double-blinded benchmarking to gold standard quantitative-RT-PCR performed by human diagnostics laboratories confirms this high sensitivity. SARSeq can be used to detect Influenza A and B viruses and human rhinovirus in parallel, and can be expanded for detection of other pathogens. Thus, SARSeq is ideally suited for differential diagnostic of infections during a pandemic.

Frozen Section of Thyroid and Parathyroid Specimens

2005 ◽  
Vol 129 (12) ◽  
pp. 1575-1584 ◽  
Author(s):  
Rose C. Anton ◽  
Thomas M. Wheeler
Keyword(s):  
Thyroid Nodules ◽  
Frozen Section ◽  
False Positive Rate ◽  
Cost Effective ◽  
Needle Aspiration ◽  
Thyroid Lobectomy ◽  
Parathyroid Lesions ◽  
Surgical Evaluation ◽  
Positive Rate ◽  
Accurate Procedure

Abstract Context.—Preoperative fine-needle aspiration of thyroid lesions has greatly diminished the need for surgical evaluation. However, because thyroid nodules are common lesions, many still require surgical intervention and represent a substantial number of cases that the pathologist encounters in the frozen section laboratory. Objective.—Comprehensive reviews of frozen section indications, as well as gross, cytologic, and histologic features of the most common and diagnostically important thyroid and parathyroid lesions, are presented to provide a guideline for proper triage and management of these cases in the frozen section laboratory. The most common pitfalls are discussed in an attempt to avoid discordant diagnoses. Data Sources.—Thyroid lobectomy, subtotal or total thyroidectomy, and parathyroid biopsy or parathyroidectomy cases are included in this review. Conclusions.—The frozen section evaluation of thyroid and parathyroid lesions remains a highly accurate procedure with a low false-positive rate. Gross inspection, complemented by cytologic and histologic review, provides the surgeon with the rapid, reliable, cost-effective information necessary for optimum patient care.

scholarly journals Clinical and Economic Evaluation after Adopting Contingent Cell-Free DNA Screening for Fetal Trisomies in South Spain

2020 ◽  
Vol 47 (10) ◽  
pp. 749-756
Author(s):  
José A. Sainz ◽  
María R. Torres ◽  
Ignacio Peral ◽  
Reyes Granell ◽  
Manuel Vargas ◽  
...  
Keyword(s):  
False Positive Rate ◽  
Cost Effective ◽  
First Trimester ◽  
Nuchal Translucency ◽  
University Hospitals ◽  
Combined Test ◽  
Positive Rate ◽  
Fetal Malformations ◽  
Cfdna Screening ◽  
Singleton Pregnancies

<b><i>Introduction:</i></b> Contingent cell-free (cf) DNA screening on the basis of the first-trimester combined test (FCT) results has emerged as a cost-effective strategy for screening of trisomy 21 (T21). <b><i>Objectives:</i></b> To assess performance, patients’ uptake, and cost of contingent cfDNA screening and to compare them with those of the established FCT. <b><i>Methods:</i></b> This is a prospective cohort study including all singleton pregnancies attending to their FCT for screening of T21 at 2 university hospitals in South Spain. When the FCT risk was ≥1:50, there were major fetal malformations, or the nuchal translucency was ≥3.5 mm, women were recommended invasive testing (IT); if the risk was between 1:50 and 1:270, women were recommended cfDNA testing; and for risks bellow 1:270, no further testing was recommended. Detection rate (DR), false-positive rate (FPR), patients’ uptake, and associated costs were evaluated. <b><i>Results:</i></b> We analyzed 10,541 women, including 46 T21 cases. DR of our contingent strategy was 89.1% (41/46) at 1.4% (146/10,541) FPR. Uptake of cfDNA testing was 91.2% (340/373), and overall IT rate was 2.0%. The total cost of our strategy was €1,462,895.7, similar to €1,446,525.7 had cfDNA testing not been available. <b><i>Conclusions:</i></b> Contingent cfDNA screening shows high DR, low IT rate, and high uptake at a similar cost than traditional screening.

scholarly journals Surveillance of Respiratory Viruses in Long Term Care Facilities

2019 ◽  
Vol 11 (1) ◽  
Author(s):  
Mary Checivich ◽  
Shari Barlow ◽  
Peter Shult ◽  
Erik Residorf ◽  
Jonathan L. Temte
Keyword(s):  
Influenza A ◽  
Nasal Swab ◽  
Long Term Care ◽  
Respiratory Infections ◽  
Respiratory Viruses ◽  
Influenza B ◽  
Rt Pcr ◽  
Term Care ◽  
Care Facilities

ObjectiveTo assess the feasibility of conducting respiratory virus surveillance for residents of long term care facilities (LTCF) using simple nasal swab specimens and to describe the virology of acute respiratory infections (ARI) in LCTFs.IntroductionAlthough residents of LTCFs have high morbidity and mortality associated with ARIs, there is very limited information on the virology of ARI in LTCFs.[1,2] Moreover, most virological testing of LCTF residents is reactive and is triggered by a resident meeting selected surveillance criteria. We report on incidental findings from a prospective trial of introducing rapid influenza diagnostic testing (RIDT) in ten Wisconsin LTCFs over a two-year period with an approach of testing any resident with ARI.MethodsAny resident with new onset of respiratory symptoms consistent with ARI had a nasal swab specimen collected for RIDT by nursing staff. Following processing for RIDT (Quidel Sofia Influenza A+B FIA), the residual swab was placed into viral transport medium and forwarded to the Wisconsin State Laboratory of Hygiene and tested for influenza using RT-PCR (IVD CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel), and for 17 viruses (Luminex NxTAG Respiratory Pathogen Panel [RPP]). The numbers of viruses in each of 7 categories [influenza A (FluA ), influenza B (FluB), coronaviruses (COR), human metapneumovirus (hMPV), parainfluenza (PARA), respiratory syncytial virus (RSV) and rhinovirus/enterovirus (R/E)], across the two years were compared using chi-square.ResultsTotals of 164 and 190 specimens were submitted during 2016-2017 and 2017-2018, respectively. RPP identified viruses in 56.2% of specimens, with no difference in capture rate between years (55.5% vs. 56.8%). Influenza A (21.5%), influenza B (16.5%), RSV (19.0%) and hMPV (16.5%) accounted for 73.5% of all detections, while coronaviruses (15.5%), rhino/enteroviruses (8.5%) and parainfluenza (2.5%) were less common. Specific distribution of viruses varied significantly across the two years (Table: X2=48.1, df=6; p<0.001).ConclusionsSurveillance in LTCFs using nasal swabs collected for RIDT is highly feasible and yields virus identification rates similar to those obtained in clinical surveillance of ARI with collection of nasopharyngeal specimens by clinicians and those obtained in a school-based surveillance project of ARI with collection of combined nasal and oropharyngeal specimens collected by trained research assistants. Significant differences in virus composition occurred across the two study years. RSV varied little between years while hMPV demonstrated wide variation. Simple approaches to surveillance may provide a more comprehensive assessment of respiratory viruses in LTCF settings.References(1) Uršič T, Gorišek Miksić N, Lusa L, Strle F, Petrovec M. Viral respiratory infections in a nursing home: a six-month prospective study. BMC Infect Dis. 2016; 16: 637. Published online 2016 Nov 4. doi: 10.1186/s12879-016-1962-8(2) Masse S, Capai L, Falchi A. Epidemiology of Respiratory Pathogens among Elderly Nursing Home Residents with Acute Respiratory Infections in Corsica, France, 2013–2017. Biomed Res Int. 2017; 2017: 1423718. Published online 2017 Dec 17. doi: 10.1155/2017/1423718

Comparing the clinical and economic effects of clinical examination, pulse oximetry, and echocardiography in newborn screening for congenital heart defects: A probabilistic cost-effectiveness model and value of information analysis

2007 ◽  
Vol 23 (2) ◽  
pp. 192-204 ◽  
Author(s):  
Ingolf Griebsch ◽  
Rachel L. Knowles ◽  
Jacqueline Brown ◽  
Catherine Bull ◽  
Christopher Wren ◽  
...  
Keyword(s):  
Cost Effectiveness ◽  
Newborn Screening ◽  
Pulse Oximetry ◽  
Clinical Examination ◽  
Heart Defects ◽  
False Positive Rate ◽  
Research Priorities ◽  
Cost Effective ◽  
Positive Rate ◽  
Clinically Significant

Objectives: Congenital heart defects (CHD) are an important cause of death and morbidity in early childhood, but the effectiveness of alternative newborn screening strategies in preventing the collapse or death—before diagnosis—of infants with treatable but life-threatening defects is uncertain. We assessed their effectiveness and efficiency to inform policy and research priorities.Methods: We compared the effectiveness of clinical examination alone and clinical examination with either pulse oximetry or screening echocardiography in making a timely diagnosis of life-threatening CHD or in diagnosing clinically significant CHD. We contrasted their cost-effectiveness, using a decision-analytic model based on 100,000 live births, and assessed future research priorities using value of information analysis.Results: Clinical examination alone, pulse oximetry, and screening echocardiography achieved 34.0, 70.6, and 71.3 timely diagnoses per 100,000 live births, respectively. This finding represents an additional cost per additional timely diagnosis of £4,894 and £4,496,666 for pulse oximetry and for screening echocardiography. The equivalent costs for clinically significant CHD are £1,489 and £36,013, respectively. Key determinants of cost-effectiveness are detection rates and screening test costs. The false-positive rate is very high with screening echocardiography (5.4 percent), but lower with pulse oximetry (1.3 percent) or clinical examination alone (.5 percent).Conclusions: Adding pulse oximetry to clinical examination is likely to be a cost-effective newborn screening strategy for CHD, but further research is required before this policy can be recommended. Screening echocardiography is unlikely to be cost-effective, unless the detection of all clinically significant CHD is considered beneficial and a 5 percent false-positive rate acceptable.

scholarly journals Follow-Up SARS-CoV-2 PCR Testing Outcomes From a Large Reference Lab in the US

2021 ◽  
Vol 9 ◽  
Author(s):  
Adam Sullivan ◽  
David Alfego ◽  
Brian Poirier ◽  
Jonathan Williams ◽  
Dorothy Adcock ◽  
...  
Keyword(s):  
United States ◽  
Cohort Study ◽  
False Positive ◽  
False Positive Rate ◽  
The United States ◽  
Positive Test ◽  
Test Results ◽  
Rt Pcr ◽  
Positive Rate

By analyzing COVID-19 sequential COVID-19 test results of patients across the United States, we herein attempt to quantify some of the observations we've made around long-term infection (and false-positive rates), as well as provide observations on the uncertainty of sampling variability and other dynamics of COVID-19 infection in the United States. Retrospective cohort study of a registry of RT-PCR testing results for all patients tested at any of the reference labs operated by Labcorp® including both positive, negative, and inconclusive results, from March 1, 2020 to January 28, 2021, including patients from all 50 states and outlying US territories. The study included 22 million patients with RT-PCR qualitative test results for SARS-CoV-2, of which 3.9 million had more than one test at Labcorp. We observed a minuscule &lt;0.1% basal positive rate for follow up tests &gt;115 days, which could account for false positives, long-haulers, and/or reinfection but is indistinguishable in the data. In observing repeat-testing, for patients who have a second test after a first RT-PCR, 30% across the cohort tested negative on the second test. For patients who test positive first and subsequently negative within 96 h (40% of positive test results), 18% of tests will subsequently test positive within another 96-h span. For those who first test negative and then positive within 96 h (2.3% of negative tests), 56% will test negative after a third and subsequent 96-h period. The sudden changes in RT-PCR test results for SARS-CoV-2 from this large cohort study suggest that negative test results during active infection or exposure can change rapidly within just days or hours. We also demonstrate that there does not appear to be a basal false positive rate among patients who test positive &gt;115 days after their first RT-PCR positive test while failing to observe any evidence of widespread reinfection.

scholarly journals Detection of Rare Drug Resistance Mutations by Digital PCR in a Human Influenza A Virus Model System and Clinical Samples

2015 ◽  
Vol 54 (2) ◽  
pp. 392-400 ◽  
Author(s):  
Alexandra S. Whale ◽  
Claire A. Bushell ◽  
Paul R. Grant ◽  
Simon Cowen ◽  
Ion Gutierrez-Aguirre ◽  
...  
Keyword(s):  
Influenza A ◽  
False Positive Rate ◽  
Digital Pcr ◽  
Clinical Samples ◽  
Nucleotide Polymorphisms ◽  
Trace Detection ◽  
Resistance Mutations ◽  
Accuracy And Precision ◽  
Positive Rate ◽  
Virus Model

Digital PCR (dPCR) is being increasingly used for the quantification of sequence variations, including single nucleotide polymorphisms (SNPs), due to its high accuracy and precision in comparison with techniques such as quantitative PCR (qPCR) and melt curve analysis. To develop and evaluate dPCR for SNP detection using DNA, RNA, and clinical samples, an influenza virus model of resistance to oseltamivir (Tamiflu) was used. First, this study was able to recognize and reduce off-target amplification in dPCR quantification, thereby enabling technical sensitivities down to 0.1% SNP abundance at a range of template concentrations, a 50-fold improvement on the qPCR assay used routinely in the clinic. Second, a method was developed for determining the false-positive rate (background) signal. Finally, comparison of dPCR with qPCR results on clinical samples demonstrated the potential impact dPCR could have on clinical research and patient management by earlier (trace) detection of rare drug-resistant sequence variants. Ultimately this could reduce the quantity of ineffective drugs taken and facilitate early switching to alternative medication when available. In the short term such methods could advance our understanding of microbial dynamics and therapeutic responses in a range of infectious diseases such as HIV, viral hepatitis, and tuberculosis. Furthermore, the findings presented here are directly relevant to other diagnostic areas, such as the detection of rare SNPs in malignancy, monitoring of graft rejection, and fetal screening.

scholarly journals Same-species Contamination Detection With Variant Calling Information From Next-generation Sequencing

2021 ◽  
Author(s):  
Tao Jiang ◽  
Martin Buchkovich ◽  
Alison Motsinger-Reif
Keyword(s):  
Quality Control ◽  
Next Generation Sequencing ◽  
False Positive ◽  
Copy Number ◽  
False Positive Rate ◽  
Variant Calling ◽  
Degraded Samples ◽  
Svm Model ◽  
Positive Rate ◽  
Generation Sequencing

Abstract Background: Same-species contamination detection is an important quality control step in genetic data analysis. Due to a scarcity of methods to detect and correct for this quality control issue, same-species contamination is more difficult to detect than cross-species contamination. We introduce a novel machine learning algorithm to detect same-species contamination in next-generation sequencing (NGS) data using a support vector machine (SVM) model. Our approach uniquely detects contamination using variant calling information stored in variant call format (VCF) files for DNA or RNA. Importantly, it can differentiate between same-species contamination and mixtures of tumor and normal cells.In the first stage, a change-point detection method is used to identify copy number variations (CNVs) and copy number aberrations (CNAs) for filtering. Next, single nucleotide polymorphism (SNP) data is used to test for same-species contamination using an SVM model. Based on the assumption that alternative allele frequencies in NGS follow the beta-binomial distribution, the deviation parameter ρ is estimated by the maximum likelihood method. All features of a radial basis function (RBF) kernel SVM are generated using publicly available or private training data. Results: We demonstrate our approach in simulation experiments. The datasets combine, in silico, exome sequencing data of DNA from two lymphoblastoid cell lines (NA12878 and NA10855). We generate VCF files using variants identified in these data and then evaluate the power and false-positive rate of our approach. Our approach can detect contamination levels as low as 5% with a reasonable false-positive rate. Results in real data have sensitivity above 99.99% and specificity of 90.24%, even in the presence of degraded samples with similar features as contaminated samples. We provide an R software implementation of our approach.Conclusions: Our approach addresses the gap in methods to test for same-species contamination in NGS. Due to its high sensitivity for degraded samples and tumor-normal samples, it represents an important tool that can be applied within the quality control process. Additionally, the user-friendly software has the unique ability to conduct quality control using the VCF format.

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