scholarly journals Validation of Saliva and Self-Administered Nasal Swabs for COVID-19 Testing

2020 ◽  
Author(s):  
Alvin Kuo Jing Teo ◽  
Yukti Choudhury ◽  
Iain Beehuat Tan ◽  
Chae Yin Cher ◽  
Shi Hao Chew ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Migrant Workers ◽  
Phylogenetic Analyses ◽  
Kappa Statistic ◽  
Sample Type ◽  
Next Generation ◽  
Rt Pcr ◽  
Male Adult ◽  
Public Health Decision ◽  
Generation Sequencing

Background Active cases of COVID-19 has primarily been diagnosed via RT-PCR of nasopharyngeal (NP) swabs. Saliva and self-administered nasal (SN) swabs can be collected safely without trained staff. We aimed to test the sensitivity of naso-oropharyngeal saliva and SN swabs compared to NP swabs in a large cohort of migrant workers in Singapore. Methods We recruited 200 male adult subjects: 45 with acute respiratory infection, 104 asymptomatic close contacts, and 51 confirmed COVID-19 cases. Each subject underwent NP swab, SN swab and saliva collection for RT-PCR testing at 1 to 3 timepoints. We additionally used a direct-from-sample amplicon-based next-generation sequencing (NGS) workflow to establish phylogeny. Results Of 200 subjects, 91 and 46 completed second and third rounds of testing, respectively. Of 337 sets of tests, there were 150 (44.5%) positive NP swabs, 127 (37.7%) positive SN swabs, and 209 (62.0%) positive saliva. Test concordance between different sample sites was good, with a kappa statistic of 0.616 for NP and SN swabs, and 0.537 for NP and saliva. In confirmed symptomatic COVID-19 subjects, the likelihood of a positive test from any sample fell beyond 14 days of symptom onset. NGS was conducted on 18 SN and saliva samples, with phylogenetic analyses demonstrating lineages for all samples tested were Clade O (GISAID nomenclature) and lineage B.6 (PANGOLIN nomenclature). Conclusion This study supports saliva as a sensitive and less intrusive sample for COVID-19 diagnosis and further delineates the role of oropharyngeal secretions in increasing the sensitivity of testing. However, SN swabs were inferior as an alternate sample type. Our study also provides evidence that a straightforward next-generation sequencing workflow can provide direct-from-sample phylogenetic analysis for public health decision-making.

scholarly journals Next-generation sequencing and RT-PCR to identify a 32-day SARS-CoV-2 carrier

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Lingjie Song ◽  
Guibao Xiao ◽  
Ruixiang Tang ◽  
Xianqin Zhang ◽  
Zhan Gao ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Next Generation ◽  
Rt Pcr ◽  
Generation Sequencing

scholarly journals SARS-CoV-2 as a Trigger in the Development of Tourette’s-Like Symptoms: A Case Report

2021 ◽  
Author(s):  
Sabine Hazan ◽  
Sheldon Jordan
Keyword(s):  
Case Report ◽  
Next Generation Sequencing ◽  
Gut Microbiota ◽  
Dramatic Improvement ◽  
Next Generation ◽  
Rt Pcr ◽  
Gastrointestinal Microbiota ◽  
Next Generation Sequencing Ngs ◽  
The Impact ◽  
Generation Sequencing

Abstract Background: Reports have been surfacing surrounding CNS-associated symptoms in individuals affected by coronavirus disease 19 (COVID-19). Tourette syndrome is a neuropsychiatric disorder with usual onset in childhood. Gut microbiota can affect central physiology and function via the microbiota-gut-brain axis. The authors of this case report describe Tourette’s-like symptoms in a patient resulting from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection disrupting gut microbiota. Case Presentation: This case involves a 16-year-old female that developed acute onset Tourette’s-like symptoms along with neuropsychiatric symptoms after exposure to and infection from SARS-CoV-2. The patient had negative nasopharyngeal (NP) real-time reverse transcription-PCR (RT-PCR) tests for SARS-CoV-2 on five occasions from August of 2020 through June of 2021. The patient’s symptoms continued to worsen over the next six months until next-generation sequencing (NGS) revealed SARS-CoV-2 in her stool. Her treatment was adjusted as NGS revealed SARS-CoV-2 in her stool. Repair of the gastrointestinal microbiota, treatment with nutraceutical and pharmaceutical agents, as well as alterations in her surroundings resulted in dramatic improvement in the microbiome and a significant reduction of symptoms.Discussion: The use of (RT-PCR) testing to determine the presence or absence of SARS-CoV-2 may be inadequate and inaccurate for individuals that have been exposed to the virus. In addition, the impact of SARS-CoV-2 infection of the GI tract may cause significant havoc in the gut microbiota. Additional testing, eradication of infectious agents, as well as restoration of the gut microbiome are needed to effectively manage and treat this condition. The patient’s symptoms worsened over the next six months until next-generation sequencing (NGS) revealed SARS-CoV-2 in her stool and her treatment was adjusted. Treatment with nutraceuticals and alterations in her surroundings was followed by a more normal microbiome and a dramatic reduction in symptoms.

scholarly journals Pediatric Brainstem Encephalitis Outbreak Investigation with Metagenomic Next-Generation Sequencing

2018 ◽  
Author(s):  
Kristoffer E. Leon ◽  
Didac Casas-Alba ◽  
Akshaya Ramesh ◽  
Lillian M. Khan ◽  
Cristian Launes ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Phylogenetic Analyses ◽  
P Value ◽  
Brainstem Encephalitis ◽  
Next Generation ◽  
Single Nucleotide Variants ◽  
Qrt Pcr ◽  
Mcnemar’S Test ◽  
Generation Sequencing ◽  
Mcnemar's Test

AbstractIn 2016, Catalonia experienced a pediatric brainstem encephalitis outbreak caused by enterovirus A71 (EV-A71). Conventional testing identified EV in peripheral body sites, but EV was rarely identified in cerebrospinal fluid (CSF). RNA was extracted from CSF (n=20), plasma (n=9), stool (n=15) and nasopharyngeal samples (n=16) from 10 children with brainstem encephalitis or encephalomyelitis and 10 contemporaneous pediatric controls with presumed viral meningitis or encephalitis. Unbiased complementary DNA libraries were sequenced, and microbial pathogens were identified using a custom bioinformatics pipeline. Full-length virus genomes were assembled for phylogenetic analyses. Metagenomic next-generation sequencing (mNGS) was concordant with qRT-PCR for all samples positive by PCR (n=25). In virus-negative samples (n=35), mNGS detected virus in 28.6% (n=10), including 5 CSF samples. mNGS co-detected EV-A71 and another EV in 5 patients. Overall, mNGS increased the proportion of EV-positive samples from 42% (25/60) to 57% (34/60) (McNemar’s test; p-value = 0.0077). For CSF, mNGS doubled the number of pathogen-positive samples (McNemar’s test; p-value = 0.074). Using phylogenetic analysis, the outbreak EV-A71 clustered with a neuroinvasive German EV-A71 isolate. Brainstem encephalitis specific, non-synonymous EV-A71 single nucleotide variants were not identified. mNGS demonstrated 100% concordance with clinical qRT-PCR of EV-related brainstem encephalitis and significantly increased the detection of enteroviruses. Our findings increase the probability that neurologic complications observed were virus-induced rather than para-infectious. A comprehensive genomic analysis confirmed that the EV-A71 outbreak strain was closely related to a neuroinvasive German EV-A71 isolate. There were no clear-cut viral genomic differences that discriminated between patients with differing neurologic phenotypes.

scholarly journals Next-Generation Sequencing-Based Detection of Common Bean Viruses in Wild Plants from Tanzania and Their Mechanical Transmission to Common Bean Plants

Plant Disease ◽  
2021 ◽  
Author(s):  
Beatrice Mwaipopo ◽  
Minna-Liisa Rajamäki ◽  
Neema Ngowi ◽  
Susan N’chimbi Msolla ◽  
P Njau ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Common Bean ◽  
Mosaic Virus ◽  
Viral Disease ◽  
Wild Plants ◽  
Next Generation ◽  
Rt Pcr ◽  
Bean Plants ◽  
Generation Sequencing ◽  
Common Bean Plants

Viral diseases are a major threat for common bean production. In recent surveys, >15 different viruses belonging to 11 genera were shown to infect common bean (Phaseolus vulgaris L.) in Tanzania. Management of viruses requires an understanding of how they survive from one season to the next. In this study, we explored the possibility that alternative host plants have a central role in the survival of common bean viruses. We used next-generation sequencing (NGS) techniques to sequence virus-derived small interfering RNAs, together with conventional reverse transcription-polymerase chain reaction (RT-PCR) to detect viruses in wild plants. Leaf samples for RNA extraction and NGS were collected from 1,430 wild plants around and within common bean fields in four agricultural zones in Tanzania. At least partial genome sequences of viruses potentially belonging to 25 genera were detected. The greatest virus diversity was detected in the eastern and northern zones, whereas wild plants in the Lake zone and especially in the southern highlands zone showed only a few viruses. RT-PCR analysis of all the collected plant samples confirmed the presence of yam bean mosaic virus and peanut mottle virus in wild legume plants. Of all viruses detected, only two viruses, cucumber mosaic virus and a novel bromovirus related to cowpea chlorotic mottle virus and brome mosaic virus, were mechanically transmitted from wild plants to common bean plants. The data generated in this study are crucial for development of viral disease management strategies and predicting crop viral disease outbreaks in different agricultural regions in Tanzania and beyond.

scholarly journals Towards Translational Epidemiology: Next-Generation Sequencing and Phylogeography as Epidemiological Mainstays

mSystems ◽  
2019 ◽  
Vol 4 (3) ◽  
Author(s):  
Crystal M. Hepp
Keyword(s):  
Public Health ◽  
Next Generation Sequencing ◽  
Phylogenetic Analyses ◽  
Federal State ◽  
Next Generation ◽  
Public Health Agencies ◽  
Health Agencies ◽  
Collaborative Activities ◽  
User Friendly ◽  
Generation Sequencing

ABSTRACT Next-generation sequencing, coupled with the development of user-friendly software, has achieved a level of accessibility that is revolutionizing the way we approach epidemiological investigations. We can sequence pathogen genomes and conduct phylogenetic analyses to assess transmission, identify from which country or city a pathogen originated, or which contaminated potluck item resulted in widespread foodborne illness. However, until recently, these types of studies have been rarities, limited to specific investigations usually conducted over the short term. Given the feasibility and realized public health benefits of ascertaining pathogen relationships, federal, state, and county agencies are building their sequencing capacities, either through acquisition of equipment or collaborative activities. In this perspective, I detail research projects that our group collaborates on with county and state public health agencies, where the objective is to identify pathogen source locations with the longer-term goal of implementing proactive interventions.

scholarly journals Recombinant GII.P16/GII.4 Sydney 2012 Was the Dominant Norovirus Identified in Australia and New Zealand in 2017

Viruses ◽  
2018 ◽  
Vol 10 (10) ◽  
pp. 548 ◽  
Author(s):  
Jennifer Lun ◽  
Joanne Hewitt ◽  
Grace Yan ◽  
Daniel Enosi Tuipulotu ◽  
William Rawlinson ◽  
...  
Keyword(s):  
New Zealand ◽  
Next Generation Sequencing ◽  
Virus Infection ◽  
Phylogenetic Analyses ◽  
Rt Pcr ◽  
Molecular Surveillance ◽  
Recombinant Viruses ◽  
The Past ◽  
Wastewater Samples ◽  
Generation Sequencing

For the past two decades, norovirus pandemic variants have emerged every 3–5 years, and dominate until they are replaced by alternate strains. However, this scenario changed in 2016 with the co-circulation of six prevalent viruses, three of which possessed the pandemic GII.4 Sydney 2012 capsid. An increased number of institutional gastroenteritis outbreaks were reported within the Oceania region in mid-2017. This study identified emerging noroviruses circulating in Australia and New Zealand in 2017 to assess the changing dynamics of the virus infection. RT-PCR-based methods, next generation sequencing, and phylogenetic analyses were used to genotype noroviruses from both clinical and wastewater samples. Antigenic changes were observed between the capsid of pandemic Sydney 2012 variant and the two new Sydney recombinant viruses. The combination of these antigenic changes and the acquisition of a new ORF1 through recombination could both facilitate their ongoing persistence in the population. Overall, an increased prevalence of GII.P16/GII.4 Sydney 2012 viruses was observed in 2017, replacing the GII.P16/GII.2 recombinant that dominated in the region at the end of 2016. This shift in strain dominance was also observed in wastewater samples, demonstrating the reliability of wastewater as a molecular surveillance tool.

scholarly journals Epidemiological data analysis of viral quasispecies in the next-generation sequencing era

2020 ◽  
Author(s):  
Sergey Knyazev ◽  
Lauren Hughes ◽  
Pavel Skums ◽  
Alexander Zelikovsky
Keyword(s):  
Next Generation Sequencing ◽  
Large Scale ◽  
Phylogenetic Analyses ◽  
Epidemiological Surveillance ◽  
Viral Population ◽  
Surveillance Systems ◽  
Next Generation ◽  
Effective Prevention ◽  
Ngs Data ◽  
Generation Sequencing

Abstract The unprecedented coverage offered by next-generation sequencing (NGS) technology has facilitated the assessment of the population complexity of intra-host RNA viral populations at an unprecedented level of detail. Consequently, analysis of NGS datasets could be used to extract and infer crucial epidemiological and biomedical information on the levels of both infected individuals and susceptible populations, thus enabling the development of more effective prevention strategies and antiviral therapeutics. Such information includes drug resistance, infection stage, transmission clusters and structures of transmission networks. However, NGS data require sophisticated analysis dealing with millions of error-prone short reads per patient. Prior to the NGS era, epidemiological and phylogenetic analyses were geared toward Sanger sequencing technology; now, they must be redesigned to handle the large-scale NGS datasets and properly model the evolution of heterogeneous rapidly mutating viral populations. Additionally, dedicated epidemiological surveillance systems require big data analytics to handle millions of reads obtained from thousands of patients for rapid outbreak investigation and management. We survey bioinformatics tools analyzing NGS data for (i) characterization of intra-host viral population complexity including single nucleotide variant and haplotype calling; (ii) downstream epidemiological analysis and inference of drug-resistant mutations, age of infection and linkage between patients; and (iii) data collection and analytics in surveillance systems for fast response and control of outbreaks.

scholarly journals Highly sensitive and full-genome interrogation of SARS-CoV-2 using multiplexed PCR enrichment followed by next-generation sequencing

2020 ◽  
Author(s):  
Chenyu Li ◽  
David N. Debruyne ◽  
Julia Spencer ◽  
Vidushi Kapoor ◽  
Lily Y. Liu ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Multiplex Pcr ◽  
High Sensitivity ◽  
Detection Methods ◽  
Next Generation ◽  
Rt Pcr ◽  
Copy Numbers ◽  
Virus Rna ◽  
Highly Sensitive ◽  
Generation Sequencing

AbstractMany detection methods have been used or reported for the diagnosis and/or surveillance of COVID-19. Among them, reverse transcription polymerase chain reaction (RT-PCR) is the most commonly used because of its high sensitivity, typically claiming detection of about 5 copies of viruses. However, it has been reported that only 47-59% of the positive cases were identified by some RT-PCR methods, probably due to low viral load, timing of sampling, degradation of virus RNA in the sampling process, or possible mutations spanning the primer binding sites. Therefore, alternative and highly sensitive methods are imperative. With the goal of improving sensitivity and accommodating various application settings, we developed a multiplex-PCR-based method comprised of 343 pairs of specific primers, and demonstrated its efficiency to detect SARS-CoV-2 at low copy numbers. The assay produced clean characteristic target peaks of defined sizes, which allowed for direct identification of positives by electrophoresis. We further amplified the entire SARS-CoV-2 genome from 8 to half a million viral copies purified from 13 COVID-19 positive specimens, and detected mutations through next generation sequencing. Finally, we developed a multiplex-PCR-based metagenomic method in parallel, that required modest sequencing depth for uncovering SARS-CoV-2 mutational diversity and potentially novel or emerging isolates.

scholarly journals Chronic SARS-CoV-2 infection and viral evolution in a hypogammaglobulinaemic individual.

2021 ◽  
Author(s):  
Maia Kavanagh Williamson ◽  
Fergus Hamilton ◽  
Stephanie Hutchings ◽  
Hannah M Pymont ◽  
Mark Hackett ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Amino Acid ◽  
Selective Pressure ◽  
Viral Evolution ◽  
Viral Escape ◽  
Spike Protein ◽  
Next Generation ◽  
Rt Pcr ◽  
The Face ◽  
Generation Sequencing

There is widespread interest in the capacity for SARS-CoV-2 evolution in the face of selective pressures from host immunity, either naturally acquired post-exposure or from vaccine acquired immunity. Allied to this is the potential for long perm persistent infections within immune compromised individuals to allow a broader range of viral evolution in the face of sub-optimal immune driven selective pressure. Here we report on an immunocompromised individual who is hypogammaglobulinaemic and was persistently infected with SARS-CoV-2 for over 290 days, the longest persistent infection recorded in the literature to date. During this time, nine samples of viral nucleic acid were obtained and analysed by next-generation sequencing. Initially only a single mutation (L179I) was detected in the spike protein relative to the prototypic SARS-CoV-2 Wuhan-Hu-1 isolate, with no further changes identified at day 58. However, by day 155 the spike protein had acquired a further four amino acid changes, namely S255F, S477N, H655Y and D1620A and a two amino acid deletion (ΔH69/ΔV70). Infectious virus was cultured from a nasopharyngeal sample taken on day 155 and next-generation sequencing confirmed that the mutations in the virus mirrored those identified by sequencing of the corresponding swab sample. The isolated virus was susceptible to remdesivir in vitro, however a 17-day course of remdesivir started on day 213 had no effect on the viral RT-PCR cycle threshold (Ct) value. On day 265 the patient was treated with the combination of casirivimab and imdevimab. The patient experienced progressive resolution of all symptoms over the next 8 weeks and by day 311 the virus was no longer detectable by RT-PCR. The ΔH69/ΔV70 deletion in the N-terminus of the spike protein which arose in our patient is also present in the B.1.1.7 variant of concern and has been associated with viral escape mutagenesis after treatment of another immunocompromised patient with convalescent plasma. Our data confirms the significance of this deletion in immunocompromised patients but illustrates it can arise independently of passive antibody transfer, suggesting the deletion may be an enabling mutation that compensates for distant changes in the spike protein that arise under selective pressure.

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