scholarly journals High-Throughput Nucleotide Resolution Predictions of Assay Limitations Increase the Reliability and Concordance of Clinical Tests

2021 ◽  
pp. 1085-1095
Author(s):  
Jonathan Bieler ◽  
Christian Pozzorini ◽  
Jessica Garcia ◽  
Alex C. Tuck ◽  
Morgane Macheret ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
False Negative ◽  
Variant Calling ◽  
Biophysical Model ◽  
Sequencing Error ◽  
Genomic Context ◽  
Negative Results ◽  
False Negative Results ◽  
Estimate Reliability ◽  
Nucleotide Resolution

PURPOSE The ability of next-generation sequencing (NGS) assays to interrogate thousands of genomic loci has revolutionized genetic testing. However, translation to the clinic is impeded by false-negative results that pose a risk to patients. In response, regulatory bodies are calling for reliability measures to be reported alongside NGS results. Existing methods to estimate reliability do not account for sample- and position-specific variability, which can be significant. Here, we report an approach that computes reliability metrics for every genomic position and sample interrogated by an NGS assay. METHODS Our approach predicts the limit of detection (LOD), the lowest reliably detectable variant fraction, by taking technical factors into account. We initially explored how LOD is affected by input material amount, library conversion rate, sequencing coverage, and sequencing error rate. This revealed that LOD depends heavily on genomic context and sample properties. Using these insights, we developed a computational approach to predict LOD on the basis of a biophysical model of the NGS workflow. We focused on targeted assays for cell-free DNA, but, in principle, this approach applies to any NGS assay. RESULTS We validated our approach by showing that it accurately predicts LOD and distinguishes reliable from unreliable results when screening 580 lung cancer samples for actionable mutations. Compared with a standard variant calling workflow, our approach avoided most false negatives and improved interassay concordance from 94% to 99%. CONCLUSION Our approach, which we name LAVA (LOD-aware variant analysis), reports the LOD for every position and sample interrogated by an NGS assay. This enables reliable results to be identified and improves the transparency and safety of genetic tests.

scholarly journals Distribution of SARS-CoV-2 PCR Cycle Threshold Values Provide Practical Insight Into Overall and Target-Specific Sensitivity Among Symptomatic Patients

2020 ◽  
Vol 154 (4) ◽  
pp. 479-485 ◽  
Author(s):  
Blake W Buchan ◽  
Jessica S Hoff ◽  
Cameron G Gmehlin ◽  
Adriana Perez ◽  
Matthew L Faron ◽  
...  
Keyword(s):  
Viral Load ◽  
Limit Of Detection ◽  
False Negative ◽  
Age Group ◽  
Rt Pcr ◽  
Patient Age ◽  
Cycle Threshold ◽  
Negative Results ◽  
Patient Âgé ◽  
False Negative Results

Abstract Objectives We examined the distribution of reverse transcription polymerase chain reaction (RT-PCR) cycle threshold (CT) values obtained from symptomatic patients being evaluated for coronavirus disease 2019 (COVID-19) to determine the proportion of specimens containing a viral load near the assay limit of detection (LoD) to gain practical insight to the risk of false-negative results. We also examined the relationship between CT value and patient age to determine any age-dependent difference in viral load or test sensitivity. Methods We collected CT values obtained from the cobas severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) assay corresponding to 1,213 combined nasopharyngeal-oropharyngeal specimens obtained from symptomatic individuals that were reported as positive or presumptive positive for SARS-CoV-2. CT values were stratified by SARS-CoV target and patient age group. Results In total, 93.3% to 98.4% of specimens demonstrated CT values greater than 3× the assay LoD, at which point false-negative results would not be expected. The mean of CT values between age groups was statistically equivalent with the exception of patients in age group 80 to 89 years, which demonstrated slightly lower CTs. Conclusions Based on the distribution of observed CT values, including the small proportion of specimens with values near the assay LoD, there is a low risk of false-negative RT-PCR results in combined nasopharyngeal-oropharyngeal specimens obtained from symptomatic individuals.

scholarly journals Recurrent SARS-CoV-2 Infection: Case Reports and Review of the Literature

2021 ◽  
Vol 2 (2) ◽  
pp. 1-4
Author(s):  
Brian Conway ◽  
Keyword(s):  
Limit Of Detection ◽  
Case Reports ◽  
False Negative ◽  
Recurrent Infection ◽  
Family Unit ◽  
Her Family ◽  
Negative Results ◽  
False Negative Results ◽  
Clinical Syndromes ◽  
Relapsing Remitting

We describe a 37-year-old woman who became infected with SARS-CoV-2. Over time, 4 other members in her family unit became infected, with 3/5 developing 2-3 separate clinical syndromes over two months. It is possible that each person had a single prolonged infection, with the literature reporting RNA detection for as long as 83 days in some cases. Syndromes of relapsing/remitting infection have also been well described. Intermittent negative RNA readings may represent “false negative” results with intermittent levels of viremia that occasionally fall below the limit of detection of the assay. An alternative explanation may be multiple episodes of infection, clearance, and re-infection within the family unit. Preliminary reports in the literature suggest onward transmission after recurrent infection in 3 reported cases. An understanding of the prevalence of cases series such as ours and their pathophysiologic and immunologic significance will improve our knowledge about SARS-CoV-2 infection and strategies to control it.

Detection of microsatellite instability in a panel of solid tumours with the Idylla MSI Test using extracted DNA

2020 ◽  
Vol 74 (1) ◽  
pp. 36-42 ◽  
Author(s):  
Adrien Pécriaux ◽  
Loetitia Favre ◽  
Julien Calderaro ◽  
Cécile Charpy ◽  
Jonathan Derman ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
False Negative ◽  
Solid Tumours ◽  
Automated System ◽  
Ovarian Carcinomas ◽  
Negative Results ◽  
False Negative Results ◽  
Large Panel ◽  
Msi Analysis

AimDuring the last few years, determination of microstatellite instability (MSI) status has become a routine part of clinical practice, essentially to detect Lynch syndrome. Recently, MSI testing has increased with the development of immunotherapy and has expanded to a large panel of solid tumours. The aim of our work was to evaluate a fully automated system developed by Biocartis, the Idylla MSI Test, which performs an MSI analysis within 150 min.MethodsA comparison between pentaplex PCR, immunohistochemistry and Idylla MSI Test was performed in 53 colorectal carcinoma samples, 7 small intestine adenocarcinomas, 15 duodenal and pancreatic adenocarcinomas, 16 gastric tumours, 15 endometrial adenocarcinomas, 5 ovarian carcinomas and 4 cases of urinary tract tumours using extracted DNA. Limit-of-detection (LOD) experiment was also done using a commercial DNA known to harbour MSI phenotype.ResultsThe overall sensitivity was 94% and the overall specificity was 100%. Two invalid and three false-negative results were observed. Our experiments showed that the amount of DNA loaded into the cartridge was decisive and should be superior to 25 ng. LOD comprised between 4% and 8%.ConclusionOverall, we have demonstrated that the Idylla MSI Test is a rapid and valid option to detect MSI phenotype which can be used in a large panel of solid tumours.

scholarly journals Lateral Flow Immunochromatography Assay for Detection of Furosemide in Slimming Health Foods

Foods ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2041
Author(s):  
Yingying Li ◽  
Haihuan Xie ◽  
Jin Wang ◽  
Xiangmei Li ◽  
Zhili Xiao ◽  
...  
Keyword(s):  
Au Nanoparticles ◽  
Limit Of Detection ◽  
False Negative ◽  
Sample Pretreatment ◽  
Lateral Flow ◽  
Initial Screening ◽  
Negative Results ◽  
False Negative Results ◽  
High Consistency ◽  
First Time

In recent years, furosemide has been found to be abused in slimming health foods. There is an urgent need for a simpler, faster method for detecting furosemide in slimming health foods. In this study, a rapid, convenient and sensitive lateral flow immunochromatography (LFIA) based on Au nanoparticles (AuNPs) was established for the first time. Under optimal conditions, the qualitative limit of detection (LOD) of the AuNPs-based LFIA was 1.0~1.2 μg/g in slimming health foods with different substrates. AuNPs-LFIA could specifically detect furosemide within 12 min (including sample pretreatment) and be read by the naked eye. The developed AuNPs-LFIA showed high consistency with liquid chromatography with tandem mass spectrometry (LC-MS/MS), and no false positive or false negative results were found in spiked slimming health foods, proving that the AuNPs-LFIA should be accurate and reliable. The AuNPs-LFIA reported here provides a serviceable analytical tool for the on-site detection and rapid initial screening of furosemide for the first time.

Critical Evaluation of Impedance Phlebography for the Diagnosis of Deep Vein Thrombosis

1974 ◽  
Vol 31 (02) ◽  
pp. 273-278
Author(s):  
Kenneth K Wu ◽  
John C Hoak ◽  
Robert W Barnes ◽  
Stuart L Frankel
Keyword(s):  
Deep Vein Thrombosis ◽  
False Positive ◽  
False Negative ◽  
Critical Evaluation ◽  
Original Method ◽  
Vein Thrombosis ◽  
Negative Results ◽  
False Negative Results ◽  
Deep Vein ◽  
Positive Results

SummaryIn order to evaluate its daily variability and reliability, impedance phlebography was performed daily or on alternate days on 61 patients with deep vein thrombosis, of whom 47 also had 125I-fibrinogen uptake tests and 22 had radiographic venography. The results showed that impedance phlebography was highly variable and poorly reliable. False positive results were noted in 8 limbs (18%) and false negative results in 3 limbs (7%). Despite its being simple, rapid and noninvasive, its clinical usefulness is doubtful when performed according to the original method.

Scoring System for the Diagnosis of COVID-19

2020 ◽  
Vol 13 (1) ◽  
pp. 413-414 ◽  
Author(s):  
Mohamed Farouk Allam
Keyword(s):  
Scoring System ◽  
Clinical Symptoms ◽  
False Negative ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Negative Results ◽  
False Negative Results ◽  
Symptoms And Signs

Due to the international spread of COVID-19, the difficulty of collecting nasopharyngeal swab specimen from all suspected patients, the costs of RT-PCR and CT, and the false negative results of RT-PCR assay in 41% of COVID-19 patients, a scoring system is needed to classify the suspected patients in order to determine the need for follow-up, home isolation, quarantine or the conduction of further investigations. A scoring system is proposed as a diagnostic tool for suspected patients. It includes Epidemiological Evidence of Exposure, Clinical Symptoms and Signs, and Investigations (if available). This scoring system is simple, could be calculated in a few minutes, and incorporates the main possible data/findings of any patient.

Detection of Streptococcus pneumoniae in Sputum Samples by Real- Time PCR.

2020 ◽  
Vol 18 ◽  
Author(s):  
Pegah Shakib ◽  
Mohammad Reza Zolfaghari
Keyword(s):  
Streptococcus Pneumoniae ◽  
Real Time ◽  
Real Time Pcr ◽  
False Negative ◽  
Cross Sectional Study ◽  
Laboratory Culture ◽  
Cross Sectional ◽  
Negative Results ◽  
False Negative Results ◽  
Pcr Method

Background: Conventional laboratory culture-based methods for diagnosis of Streptococcus pneumoniae are time-consuming and yield false negative results. Molecular methods including real-time (RT)-PCR rapid methods and conventional PCR due to higher sensitivity and accuracy have been replaced instead traditional culture assay. The aim of the current study was to evaluate lytA gene for detection of Streptococcus pneumoniae in the cerebrospinal fluid of human patients with meningitis using real-time PCR assay. Material and Methods: In this cross-sectional study, a total of 30 clinical specimens were collected from patients in a period from September to December 2018. In order to evaluate the presence of lytA gene, conventional and real-time PCR methods were used without culture. Results: From 30 sputum samples five (16.66%) isolates were identified as S. pneumoniae by lytA PCR and sequencing. Discussion: In this research, an accurate and rapid real-time PCR method was used, which is based on lytA gene for diagnosis of bacteria so that it can be diagnosed. Based on the sequencing results, the sensitivity for detection of lytA gene was 100% (5/5).

COVID-19: how nuclear medicine can provide a differential diagnosis in very dubious case

Coronaviruses ◽  
2020 ◽  
Vol 01 ◽  
Author(s):  
Maria Silvia De Feo ◽  
Viviana Frantellizzi ◽  
Giuseppe De Vincentis
Keyword(s):  
Infectious Disease ◽  
Differential Diagnosis ◽  
Ct Scan ◽  
Imaging Technique ◽  
False Negative ◽  
Clinical Signs ◽  
Negative Results ◽  
Infectious Disease Department ◽  
False Negative Results ◽  
99Mtc Hmpao

Background: We present the case of a 55-year-old woman, admitted to the Infectious Disease Department of Policlinico Umberto I, Rome, in mid-March 2020, with suspicion of COVID-19 infection. Objective: The rRT-PCR was negative and the following CT scan, performed to exclude false-negative results and help diagnosis, was inconclusive. Methods: It was decided to submit the patient to 99mTc-HMPAO-labelled leukocyte scan. Results: This exam led to the diagnosis of infective endocarditis. Conclusion: In the present pandemic scenario, 99mTc-HMPAO-labelled leukocyte scan represents a reliable imaging technique for differential diagnosis with COVID-19 in patients with confusing clinical signs, possible false-negative rRT-PCR results and inconclusive CT scan.

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