scholarly journals Amplicon-Based Detection and Sequencing of SARS-CoV-2 in Nasopharyngeal Swabs from Patients With COVID-19 and Identification of Deletions in the Viral Genome That Encode Proteins Involved in Interferon Antagonism

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1164
Author(s):  
Shona C. Moore ◽  
Rebekah Penrice-Randal ◽  
Muhannad Alruwaili ◽  
Nadine Randle ◽  
Stuart Armstrong ◽  
...  
Keyword(s):  
Viral Genome ◽  
False Negative ◽  
Point Mutations ◽  
Read Length ◽  
Diagnostic Tools ◽  
Clinical Samples ◽  
Nucleotide Polymorphisms ◽  
Pathogenic Potential ◽  
Number Of Patients ◽  
Interferon Antagonism

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Sequencing the viral genome as the outbreak progresses is important, particularly in the identification of emerging isolates with different pathogenic potential and to identify whether nucleotide changes in the genome will impair clinical diagnostic tools such as real-time PCR assays. Although single nucleotide polymorphisms and point mutations occur during the replication of coronaviruses, one of the biggest drivers in genetic change is recombination. This can manifest itself in insertions and/or deletions in the viral genome. Therefore, sequencing strategies that underpin molecular epidemiology and inform virus biology in patients should take these factors into account. A long amplicon/read length-based RT-PCR sequencing approach focused on the Oxford Nanopore MinION/GridION platforms was developed to identify and sequence the SARS-CoV-2 genome in samples from patients with or suspected of COVID-19. The protocol, termed Rapid Sequencing Long Amplicons (RSLAs) used random primers to generate cDNA from RNA purified from a sample from a patient, followed by single or multiplex PCRs to generate longer amplicons of the viral genome. The base protocol was used to identify SARS-CoV-2 in a variety of clinical samples and proved sensitive in identifying viral RNA in samples from patients that had been declared negative using other nucleic acid-based assays (false negative). Sequencing the amplicons revealed that a number of patients had a proportion of viral genomes with deletions.

scholarly journals Solid-Phase Amplification for Detection of C282Y and H63D Hemochromatosis (HFE) Gene Mutations

2001 ◽  
Vol 47 (8) ◽  
pp. 1384-1389 ◽  
Author(s):  
Mark S Turner ◽  
Sarah Penning ◽  
Angela Sharp ◽  
Valentine J Hyland ◽  
Ray Harris ◽  
...  
Keyword(s):  
Solid Phase ◽  
Point Mutations ◽  
Gene Mutations ◽  
Pcr Primers ◽  
Clinical Samples ◽  
Hfe Gene ◽  
Target Sequence ◽  
Nucleotide Polymorphisms ◽  
Nitrophenyl Phosphate ◽  
Hemochromatosis Gene

Abstract Background: There is a need for simple, rapid, and inexpensive methods for the detection of single-nucleotide polymorphisms. Our aim was to develop a single-tube ELISA-like PCR assay and evaluate it by detecting the common C282Y and H63D mutations found in the hemochromatosis gene (HFE) by use of clinical samples. Methods: The method, termed solid-phase amplification (SPA), involves dual liquid- and solid-phase amplification of a target sequence by the use of two PCR primers, one of which is in two forms: the first is covalently immobilized to the wall of a microwell, and the second is free in solution. During allele-specific amplification, both the free and solid-phase amplicons are labeled by incorporation of digoxigenin (DIG)-dUTP. The amount of surface-bound amplicon is determined colorimetrically by the use of an alkaline phosphatase-anti-DIG-Fab conjugate and p-nitrophenyl phosphate. Results: Two different amplicon-labeling methods were evaluated. Analysis of 173 clinical samples for the C282Y and H63D HFE point mutations with SPA revealed that only one sample was incorrectly diagnosed, apparently because of operator error, when compared with conventional restriction fragment length polymorphism assay results. Conclusions: The SPA assay has potential for medium-scale mutation detection, having the advantage of being manipulatively simple and immediately adaptable for use in clinical laboratories with existing ELISA instrumentation.

scholarly journals Reoccurring Bovine Anthrax in Germany on the Same Pasture after 12 Years

2021 ◽  
Author(s):  
Peter Braun ◽  
Wolfgang Beyer ◽  
Matthias Hanczaruk ◽  
Julia Riehm ◽  
Markus Antwerpen ◽  
...  
Keyword(s):  
Soil Samples ◽  
Diagnostic Tools ◽  
Clinical Samples ◽  
Nucleotide Polymorphisms ◽  
Environmental Isolates ◽  
Single Nucleotide ◽  
Virulence Plasmids ◽  
Bacterium Bacillus ◽  
Phage Receptor ◽  
Contaminated Area

The zoonotic disease anthrax caused by the endospore-forming bacterium Bacillus anthracis is very rare in Germany. In the state of Bavaria, the last case occurred in July of 2009 resulting in four dead cows. In August of 2021, the disease reemerged after heavy rains, killing one gestating cow. Notably, both outbreaks affected the same pasture, suggesting a close epidemiological connection. B. anthracis could be grown from blood culture and the presence of both virulence plasmids (pXO1 and pXO2) were confirmed by PCR. Also, recently developed diagnostic tools enabled rapid detection of B. anthracis cells and nucleic acids directly in clinical samples. The complete genome of the strain isolated from blood, designated BF-5, was DNA-sequenced and phylogenetically grouped within the B.Br.CNEVA clade that is typical for European B. anthracis strains. The genome was almost identical to BF-1, the isolate of 2009, separated only by three single nucleotide polymorphisms on the chromosome, one on plasmid pXO2 and three indel-regions. Further, B. anthracis DNA was detected by PCR from soil-samples taken from spots, where the cow had fallen onto the pasture. New tools based on phage receptor binding proteins enabled the microscopic detection and isolation of B. anthracis directly from soil-samples. These environmental isolates were genotyped and found to be SNP-identical to BF-1. Therefore, it seems that the BF-5 genotype is currently the prevalent one at the affected premises. The contaminated area was subsequently disinfected with formaldehyde.

scholarly journals Lineage-Specific Expansion of Plasmodium falciparum Parasites With pfhrp2 Deletion in the Greater Mekong Subregion

2020 ◽  
Vol 222 (9) ◽  
pp. 1561-1569
Author(s):  
Justin Gibbons ◽  
Junling Qin ◽  
Pallavi Malla ◽  
Zenglei Wang ◽  
Awtum Brashear ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
False Negative ◽  
Clinical Samples ◽  
Nucleotide Polymorphisms ◽  
Gene Deletions ◽  
Genome Wide ◽  
Lineage Specific Expansion ◽  
Specific Expansion ◽  
Survey Results ◽  
Greater Mekong Subregion

Abstract Deletion of the pfhrp2 gene in Plasmodium falciparum can lead to false-negative rapid diagnostic test (RDT) results, constituting a major challenge for evidence-based malaria treatment. Here we analyzed the whole genome sequences of 138 P. falciparum clinical samples collected from the China-Myanmar boarder for pfhrp2 and pfhrp3 gene deletions. We found pfhrp2 and pfhrp3 deletions in 9.4% and 3.6% of samples, respectively, with no samples harboring deletions of both genes. The pfhrp2 deletions showed 2 distinct breakpoints, representing 2 different chromosomal deletion events. A phylogenetic analysis performed using genome-wide single-nucleotide polymorphisms revealed that the 2 pfhrp2 breakpoint groups as well as all the pfhrp3-negative parasites formed separate clades, suggesting they might have resulted from clonal expansion of pfhrp2- and pfhrp3-negative parasites. These findings highlight the need for urgent surveys to determine the prevalence of pfhrp2-negative parasites causing false-negative RDT results and a plan for switching of RDTs pending the survey results.

scholarly journals Laboratory Diagnostic Tools for Syphilis: Current Status and Future Prospects

2021 ◽  
Vol 10 ◽  
Author(s):  
Yuting Luo ◽  
Yafeng Xie ◽  
Yongjian Xiao
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acid Amplification ◽  
Detection Methods ◽  
Current Status ◽  
Morphological Observation ◽  
Diagnostic Tools ◽  
Clinical Samples ◽  
Serologic Tests ◽  
Number Of Patients ◽  
Infectivity Test

With the increasing number of patients infected with syphilis in the past 20 years, early diagnosis and early treatment are essential to decline syphilis prevalence. Owing to its diverse manifestations, which may occur in other infections, the disease often makes clinicians confused. Therefore, a sensitive method for detecting T. pallidum is fundamental for the prompt diagnosis of syphilis. Morphological observation, immunohistochemical assay, rabbit infectivity test, serologic tests, and nucleic acid amplification assays have been applied to the diagnosis of syphilis. Morphological observation, including dark-field microscopy, silver-staining, and direct fluorescent antibody staining for T. pallidum, can be used as a direct detection method for chancre specimens in primary syphilis. Immunohistochemistry is a highly sensitive and specific assay, especially in the lesion biopsies from secondary syphilis. Rabbit infectivity test is considered as a sensitive and reliable method for detecting T. pallidum in clinical samples and used as a historical standard for the diagnosis of syphilis. Serologic tests for syphilis are widely adopted using non-treponemal or treponemal tests by either the traditional or reverse algorithm and remain the gold standard in the diagnosis of syphilis patients. In addition, nucleic acid amplification assay is capable of detecting T. pallidum DNA in the samples from patients with syphilis. Notably, PCR is probably a promising method but remains to be further improved. All of the methods mentioned above play important roles in various stages of syphilis. This review aims to provide a summary of the performance characteristics of detection methods for syphilis.

New Trends and Developments in Patients with Severe Aortic Stenosis – Towards Optimal Treatment?

2009 ◽  
Vol 5 (2) ◽  
pp. 81 ◽  
Author(s):  
Martijn WA van Geldorp ◽  
Johanna JM Takkenberg ◽  
Ad JJC Bogers ◽  
A Pieter Kappetein ◽  
◽  
...  
Keyword(s):  
Aortic Stenosis ◽  
Severe Aortic Stenosis ◽  
Surgical Techniques ◽  
Treatment Strategies ◽  
Tissue Doppler ◽  
Doppler Imaging ◽  
Treatment Modalities ◽  
Diagnostic Tools ◽  
Number Of Patients ◽  
Transcatheter Valve

Over the next few decades the number of patients diagnosed with aortic stenosis is expected to rise as the population ages and the use of several diagnostic tools expands. This will result in a growing need for both medical and surgical treatment and stimulate the development of new diagnostic and surgical techniques. This article briefly describes the prevalence, pathogenesis and clinical presentation of patients with aortic stenosis and focuses on developments in diagnostic tools, treatment strategies and treatment modalities: the use of echocardiography, tissue Doppler imaging, stress testing and biomarkers is discussed, as well as timing of surgery and the role microsimulation can play in prosthesis selection. Furthermore, newly developed transcatheter valve implantation techniques and their possible role in treating ‘inoperable’ or ‘elderly’ patients are discussed.

DETECTING MYCOBACTERIUM TUBERCULOSIS RAPIDLY AND MYCOBACTERIUM TUBERCULOSIS STRAINS WITHOUT IS6110 SEQUENCE BY PCR ASSAY

2012 ◽  
pp. 15-19
Author(s):  
Thi Chau Anh Nguyen ◽  
Hoang Bach Nguyen ◽  
Hai Duong Huynh ◽  
Nu Xuan Thanh Le ◽  
Xuan Cuong Le ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
16S Rdna ◽  
False Negative ◽  
Clinical Samples ◽  
Tuberculosis Complex ◽  
Pcr Assay ◽  
Complex Objects ◽  
Method Performance ◽  
Gene Coding ◽  
16S Rdna Pcr

Background: The Nested IS6110 PCR is used for detecting tuberculosis, however IS6110 sequence is not present in the genome of all strains of M.tuberculosis, the result may be false negative. The gene coding 16S ribosome always contains a short sequence specific to M. tuberculosis complex. Objects: Performance of the 16S Real-time PCR to detect M. tuberculosis and combining to the nested IS6110 PCR to determine the rate of Mtb strains without IS6110 from clinical samples. Materials and method: Performance of 16S rDNA PCR by commercial kit of Viet A Inc. for all 480 samples, the samples which were positive with the 16S rDNA PCR were retested in IS6110 PCR assay by in-house kit. Results: The Realtime 16S rDNA PCR detected 258 cases (53.8%) of tuberculosis. There were 3 (1.2 %) M. tuberculosis strains which do not harbor IS6110 sequence in genome. Conclusion: The IS6110 nested PCR can be applied more widely than the 16S rDNA realtime PCR. In case of using IS6110 PCR assay, results may show a low proportion of false negative. Combining 16S rDNA PCR with the IS6110 based PCR allowed detection of deletion of IS6110 sequence in M. tuberculosis isolates.

Mosquito and Tick-borne Illnesses in the United States. Guidelines for the Recognition and Empiric Treatment of Zoonotic Diseases in the Wilderness.

2019 ◽  
Vol 19 (3) ◽  
pp. 238-257
Author(s):  
Suresh Antony
Keyword(s):  
United States ◽  
Lyme Disease ◽  
Spotted Fever ◽  
Disease Process ◽  
Rocky Mountain ◽  
The United States ◽  
Health Care Facilities ◽  
Diagnostic Tools ◽  
Rocky Mountain Spotted Fever ◽  
Number Of Patients

Background:In the United States, tick-borne illnesses account for a significant number of patients that have been seen and treated by health care facilities. This in turn, has resulted in a significant morbidity and mortality and economic costs to the country.Methods:The distribution of these illnesses is geographically variable and is related to the climate as well. Many of these illnesses can be diagnosed and treated successfully, if recognized and started on appropriate antimicrobial therapy early in the disease process. Patient with illnesses such as Lyme disease, Wet Nile illness can result in chronic debilitating diseases if not recognized early and treated.Conclusion:This paper covers illnesses such as Lyme disease, West Nile illness, Rocky Mountain Spotted fever, Ehrlichia, Tularemia, typhus, mosquito borne illnesses such as enteroviruses, arboviruses as well as arthropod and rodent borne virus infections as well. It covers the epidemiology, clinical features and diagnostic tools needed to make the diagnosis and treat these patients as well.

scholarly journals 423. SARS-CoV-2 NGS Assay Powered by Biotia COVID-DX Software

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S278-S279
Author(s):  
Dorottya Nagy-Szakal ◽  
Mara Couto-Rodriguez ◽  
Joseph Barrows ◽  
Heather L Wells ◽  
Marilyne Debieu ◽  
...  
Keyword(s):  
Genetic Variants ◽  
Viral Genome ◽  
Board Member ◽  
Limit Of Detection ◽  
Clinical Samples ◽  
Analysis Tool ◽  
Viral Titer ◽  
Target Enrichment ◽  
Library Preparation ◽  
Software Analysis

Abstract Background COVID-19 had spread quickly, causing an international public health emergency with an alarming global shortage of COVID-19 diagnostic tests. We developed and clinically validated a next-generation sequencing (NGS)-based target enrichment assay with the COVID-DX Software tailored for the detection, characterization, and surveillance of the SARS-CoV-2 viral genome. Methods The SARS-CoV-2 NGS assay consists of components including library preparation, target enrichment, sequencing, and a COVID-DX Software analysis tool. The NGS library preparation starts with extracted RNA from nasopharyngeal (NP) swabs followed by cDNA synthesis and conversion to Illumina TruSeq-compatible libraries using the Twist Library Preparation Kit via Enzymatic Fragmentation and Unique Dual Indices (UDI). The library is then enriched for SARS-CoV-2 sequences using a panel of dsDNA biotin-labeled probes, specifically designed to target the SARS-CoV-2 genome, then sequenced on an Illumina NextSeq 550 platform. The COVID-DX Software analyzes sequence results and provides a clinically oriented report, including the presence/absence of SARS-CoV-2 for diagnostic use. An additional research use only report describes the assay performance, estimated viral titer, coverage across the viral genome, genetic variants, and phylogenetic analysis. Results The SARS-CoV-2 NGS Assay was validated on 30 positive and 30 negative clinical samples. To measure the sensitivity and specificity of the assay, the positive and negative percent agreement (PPA, NPA) was defined in comparison to an orthogonal EUA RT-PCR assay (PPA [95% CI]: 96.77% [90.56%-100%] and NPA [95% CI]: 100% [100%-100%]). Data reported using our assay defined the limit of detection to be 40 copies/ml using heat-inactivated SARS-CoV-2 viral genome in clinical matrices. In-silico analysis provided >99.9% coverage across the SARS-CoV-2 viral genome and no cross-reactivity with evolutionarily similar respiratory pathogens. Conclusion The SARS-CoV-2 NGS Assay powered by the COVID-DX Software can be used to detect the SARS-CoV-2 virus and provide additional insight into viral titer and genetic variants to track transmission, stratify risk, predict outcome and therapeutic response, and control the spread of infectious disease. Disclosures Dorottya Nagy-Szakal, MD PhD, Biotia (Employee) Mara Couto-Rodriguez, MS, Biotia (Employee) Joseph Barrows, MS, Biotia, Inc. (Employee, Shareholder) Heather L. Wells, MPH, Biotia (Consultant) Marilyne Debieu, PhD, Biotia (Employee) Courteny Hager, BS, Biotia (Employee) Kristin Butcher, MS, Twist Bioscience (Employee) Siyuan Chen, PhD, Twist Bioscience (Employee) Christopher Mason, PhD, Biotia (Board Member, Employee, Shareholder) Niamh B. O’Hara, PhD, Biotia (Board Member, Employee, Shareholder)Twist (Other Financial or Material Support, I am CEO of Biotia and Biotia has business partnership with Twist)

scholarly journals T stage-dependent lymph node and distant metastasis and the accuracy of lymph node assessment in rectal cancer

2021 ◽  
Author(s):  
Henry Ptok ◽  
Frank Meyer ◽  
Roland S. Croner ◽  
Ingo Gastinger ◽  
Benjamin Garlipp
Keyword(s):  
Rectal Cancer ◽  
Overall Survival ◽  
Lymph Node ◽  
Lymph Nodes ◽  
False Negative ◽  
Cumulative Risk ◽  
Distant Metastases ◽  
Number Of Patients ◽  
Tumor Stages ◽  
The Impact

Summary Objective To analyze data obtained in a representative number of patients with primary rectal cancer with respect to lymph node diagnostics and related tumor stages. Methods In pT2-, pT3-, and pT4 rectal cancer lesions, the impact of investigated lymph nodes on the frequency of pN+ status, the cumulative risk of metachronous distant metastases, and overall survival was studied by means of a prospective multicenter observational study over a defined period of time. Results From 2000 to 2011, the proportion of surgical specimens with ≥ 12 investigated lymph nodes increased significantly, from 73.6% to 93.2% (p < 0.001; the number of investigated lymph nodes from 16.2 to 20.8; p < 0.001). Despite this, the percentage of pN+ rectal cancer lesions varied only non-significantly (39.9% to 45.9%; p = 0.130; median, 44.1%). For pT2-, pT3-, and pT4 rectal cancer lesions, there was an increasing proportion of pN+ findings correlating significantly with the number of investigated lymph nodes up to n = 12 investigated lymph nodes. Only in pT3 rectal cancer was there a significant increase in pN+ findings in case of > 12 lymph nodes (p = 0.001), but not in pT2 (p = 0.655) and pT4 cancer lesions (p = 0.256). For pT3pN0cM0 rectal cancer, the risk of metachronous distant metastases and overall survival did not depend on the number of investigated lymph nodes. Conclusion In rectal cancer, at least n = 12 lymph nodes are to be minimally investigated. The investigation of fewer lymph nodes is associated with a higher risk of false-negative pN0 findings. In particular, in pT3 rectal cancer, the investigation of more than 12 lymph nodes lowers the risk of false-negative pN0 findings. An upstaging effect by the investigation of a possibly maximal number of lymph nodes could not be detected.

scholarly journals Superiority of MALDI-TOF Mass Spectrometry over Real-Time PCR for SARS-CoV-2 RNA Detection

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 730
Author(s):  
Magda Rybicka ◽  
Ewa Miłosz ◽  
Krzysztof Piotr Bielawski
Keyword(s):  
Mass Spectrometry ◽  
Gold Standard ◽  
Viral Genome ◽  
False Negative ◽  
Rt Pcr ◽  
Rna Detection ◽  
Maldi Tof ◽  
Negative Results ◽  
False Negative Results ◽  
Pcr Test

At present, the RT-PCR test remains the gold standard for early diagnosis of SARS-CoV-2. Nevertheless, there is growing evidence demonstrating that this technique may generate false-negative results. Here, we aimed to compare the new mass spectrometry-based assay MassARRAY® SARS-CoV-2 Panel with the RT-PCR diagnostic test approved for clinical use. The study group consisted of 168 suspected patients with symptoms of a respiratory infection. After simultaneous analysis by RT-PCR and mass spectrometry methods, we obtained discordant results for 17 samples (10.12%). Within fifteen samples officially reported as presumptive positive, 13 were positive according to the MS-based assay. Moreover, four samples reported by the officially approved RT-PCR as negative were positive in at least one MS assay. We have successfully demonstrated superior sensitivity of the MS-based assay in SARS-CoV-2 detection, showing that MALDI-TOF MS seems to be ideal for the detection as well as discrimination of mutations within the viral genome.

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