Saliva is more sensitive than nasopharyngeal or nasal swabs for diagnosis of asymptomatic and mild COVID-19 infection

AbstractWe aimed to test the sensitivity of naso-oropharyngeal saliva and self-administered nasal (SN) swab compared to nasopharyngeal (NP) swab for COVID-19 testing in a large cohort of migrant workers in Singapore. We also tested the utility of next-generation sequencing (NGS) for diagnosis of COVID-19. Saliva, NP and SN swabs were collected from subjects who presented with acute respiratory infection, their asymptomatic roommates, and prior confirmed cases who were undergoing isolation at a community care facility in June 2020. All samples were tested using RT-PCR. SARS-CoV-2 amplicon-based NGS with phylogenetic analysis was done for 30 samples. We recruited 200 subjects, of which 91 and 46 were tested twice and thrice respectively. In total, 62.0%, 44.5%, and 37.7% of saliva, NP and SN samples were positive. Cycle threshold (Ct) values were lower during the earlier period of infection across all sample types. The percentage of test-positive saliva was higher than NP and SN swabs. We found a strong correlation between viral genome coverage by NGS and Ct values for SARS-CoV-2. Phylogenetic analyses revealed Clade O and lineage B.6 known to be circulating in Singapore. We found saliva to be a sensitive and viable sample for COVID-19 diagnosis.

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

10.1101/2020.08.13.20173807 ◽

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.

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Detection of SARS-CoV-2 from patient fecal samples by whole genome sequencing

Gut Pathogens ◽

10.1186/s13099-021-00398-5 ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

Andreas Papoutsis ◽

Thomas Borody ◽

Siba Dolai ◽

Jordan Daniels ◽

Skylar Steinberg ◽

...

Keyword(s):

Next Generation Sequencing ◽

Nasopharyngeal Swab ◽

Whole Genome ◽

Rt Pcr ◽

Whole Genome Analysis ◽

Fecal Samples ◽

Oral Transmission ◽

Study Participants ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

Abstract Background SARS-CoV-2 has been detected not only in respiratory secretions, but also in stool collections. Here were sought to identify SARS-CoV-2 by enrichment next-generation sequencing (NGS) from fecal samples, and to utilize whole genome analysis to characterize SARS-CoV-2 mutational variations in COVID-19 patients. Results Study participants underwent testing for SARS-CoV-2 from fecal samples by whole genome enrichment NGS (n = 14), and RT-PCR nasopharyngeal swab analysis (n = 12). The concordance of SARS-CoV-2 detection by enrichment NGS from stools with RT-PCR nasopharyngeal analysis was 100%. Unique variants were identified in four patients, with a total of 33 different mutations among those in which SARS-CoV-2 was detected by whole genome enrichment NGS. Conclusion These results highlight the potential viability of SARS-CoV-2 in feces, its ongoing mutational accumulation, and its possible role in fecal–oral transmission. This study also elucidates the advantages of SARS-CoV-2 enrichment NGS, which may be a key methodology to document complete viral eradication. Trial registration ClinicalTrials.gov, NCT04359836, Registered 24 April 2020, https://clinicaltrials.gov/ct2/show/NCT04359836?term=NCT04359836&draw=2&rank=1).

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

10.21203/rs.3.rs-1079406/v1 ◽

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.

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Characterization and Comparative Genomics Analysis of two Bacillus megaterium lytic bacteriophages

10.7287/peerj.preprints.27171v1 ◽

2018 ◽

Author(s):

Abdoallah Sharaf ◽

Miroslav Obornik ◽

Adel Hammad ◽

Sohair El-Afifi ◽

Eman Marei

Keyword(s):

Bacillus Megaterium ◽

Burst Size ◽

Phylogenetic Analyses ◽

Modular Organization ◽

Environmental Diversity ◽

Egyptian Soil ◽

Comparative Genomics Analysis ◽

Next Generation Sequencing Ngs ◽

Growth Analyses ◽

Generation Sequencing

Next Generation Sequencing (NGS) technologies provide unique possibilities for the comprehensive assessment of the environmental diversity of bacteriophages. Several Bacillus bacteriophages have been isolated, but very few Bacillus megaterium bacteriophages have been characterized. In this study, we describe the biological characteristics, whole genome sequences, and their annotations for two new isolates of the B. megaterium bacteriophages (BM5 and BM10), which were isolated from Egyptian soil samples. Growth analyses indicated that the phages BM5 and BM10 have a shorter latent period (25 and 30 minutes respectively) and a smaller burst size (103 and 117 PFU respectively), in comparison to that which is typical for Bacillus phages. The genome sizes of the phages BM5 and BM10 were 165,031 bp and 165,213 bp, respectively, with a modular organization. Bioinformatic analyses of these genomes enabled the assignment of putative functions to 97 and 65 putative ORFs, respectively. Comparative analysis of the BM5 and BM10 genome structures, in conjunction with other B. megaterium bacteriophages, revealed relatively high levels of sequence and organizational identity. Both genomic comparisons and phylogenetic analyses support the conclusion that the sequenced phages (BM5 and BM10) belong to different sub-clusters (L5 and L7 respectively), within the L-cluster, and display different lifestyles (lysogenic and lytic respectively). Moreover, sequenced phages encode proteins associated with Bacillus pathogenesis. In addition, BM5 does not contain any tRNA sequences, whereas BM10 genome codes for 17 tRNAs.

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Molecular epidemiology and clinical characteristics of hepatitis delta virus (HDV) infected patients with elevated transaminases in Shanghai, China

10.21203/rs.2.22573/v1 ◽

2020 ◽

Author(s):

Shanshan Wu ◽

Yi Zhang ◽

Yuyan Tang ◽

Ting Yao ◽

Mengjiao Lv ◽

...

Keyword(s):

Next Generation Sequencing ◽

Hepatitis Delta Virus ◽

Clinical Characteristics ◽

Phylogenetic Analyses ◽

Serum Samples ◽

Next Generation Sequencing Ngs ◽

Elevated Transaminases ◽

Generation Sequencing ◽

Hdv Genotype ◽

Delta Virus

Abstract Background: Patients coinfected with HBV and hepatitis D virus (HDV) have a greater risk of HCC and cirrhosis. The current study was undertaken to assess HDV genotype distribution and determine clinical characteristics of hepatitis delta virus (HDV) among HBsAg positive individuals in Shanghai.Method: This retrospective study involved 225 serum samples from HBsAg positive hospitalized patients from October 2010 to April 2013. HDV-specific RT-nested PCR was used to amplify HDV RNA. HDV genotypes were characterized by Next-generation sequencing (NGS), followed by phylogenetic analyses. HDV/HBV co-infected patients and HBV mono-infected patients were compared clinically and virologically.Results: Out of the 225 HBsAg-positive serum samples with elevated transaminases, HDV-RNA was identified in 11 (4.9%) HBsAg positive patients. The HBV loads in the HDV positive group were significantly lower than the HDV negative HBV-infected patients. The aminotransferase enzymes were significantly higher in HDV/HBV co-infected compared to HDV negative patients (P<0.05). Phylogenetic analyses indicated that HDV-2 genotype being the predominant genotype, other HDV genotypes were not observed. HDV/HBV patients were significantly associated with a rather unfavourable clinical outcomeConclusion： In summary, our study showed that the prevalence of HDV infection in patients with elevated transaminases is not low and the predominance of HDV genotype 2 infection in Shanghai. This finding helps us to better understand the correlation of HDV/HBV co-infection. Moreover, Next-generation sequencing (NGS) technologies provide a rapid, precise method for generating HDV genomes to define infecting genotypes.

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Molecular epidemiology and clinical characteristics of hepatitis delta virus (HDV) infected patients with elevated transaminases in Shanghai, China

10.21203/rs.2.22573/v2 ◽

2020 ◽

Author(s):

Shanshan Wu ◽

Yi Zhang ◽

Yuyan Tang ◽

Ting Yao ◽

Mengjiao Lv ◽

...

Keyword(s):

Next Generation Sequencing ◽

Hepatitis Delta Virus ◽

Clinical Characteristics ◽

Phylogenetic Analyses ◽

Serum Samples ◽

Next Generation Sequencing Ngs ◽

Elevated Transaminases ◽

Generation Sequencing ◽

Hdv Genotype ◽

Delta Virus

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

Viruses ◽

10.3390/v10100548 ◽

2018 ◽

Vol 10 (10) ◽

pp. 548 ◽

Cited By ~ 10

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.

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Novel RGAG1-BCOR gene fusion revealed in a somatic soft tissue sarcoma with a long follow-up

Virchows Archiv ◽

10.1007/s00428-021-03160-z ◽

2021 ◽

Author(s):

Mauro Vasella ◽

Ulrich Wagner ◽

Christine Fritz ◽

Kati Seidl ◽

Luca Giudici ◽

...

Keyword(s):

Soft Tissue ◽

Gene Fusion ◽

Soft Tissue Mass ◽

Soft Tissue Tumors ◽

Rt Pcr ◽

Tissue Mass ◽

Myxoid Chondrosarcoma ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

AbstractBCOR-rearranged sarcomas are rare and belong to the Ewing-like sarcomas (ELS). Their morphology and histopathological features make the diagnosis challenging. We present a case, initially diagnosed as an unusual extraskeletal myxoid chondrosarcoma (EMC). A 54-year-old male patient developed an asymptomatic swelling of the lower leg. Imaging showed a 9.5-cm large intramuscular soft tissue mass. Due to its morphological and immunohistochemical profile on biopsy, it was initially diagnosed as an EMC. The patient was treated by complete resection and adjuvant radiotherapy and remained free of tumor at 7 years follow-up. Using next-generation sequencing (NGS), we retrospectively identified RGAG1-BCOR gene fusion (confirmed by RT-PCR), which has not been described in somatic soft tissue tumors so far. This finding broadens the spectrum of partner genes in the BCOR-rearranged sarcomas in a tumor with a well-documented, long clinical follow-up.

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Characterization and comparative genomic analysis of virulent and temperateBacillus megateriumbacteriophages

PeerJ ◽

10.7717/peerj.5687 ◽

2018 ◽

Vol 6 ◽

pp. e5687 ◽

Cited By ~ 2

Author(s):

Abdoallah Sharaf ◽

Miroslav Oborník ◽

Adel Hammad ◽

Sohair El-Afifi ◽

Eman Marei

Keyword(s):

Burst Size ◽

Phylogenetic Analyses ◽

Genomic Analysis ◽

Modular Organization ◽

Comparative Genomic ◽

Environmental Diversity ◽

Egyptian Soil ◽

Next Generation Sequencing Ngs ◽

Growth Analyses ◽

Generation Sequencing

Next-Generation Sequencing (NGS) technologies provide unique possibilities for the comprehensive assessment of the environmental diversity of bacteriophages. SeveralBacillusbacteriophages have been isolated, but very fewBacillus megateriumbacteriophages have been characterized. In this study, we describe the biological characteristics, whole genome sequences, and annotations for two new isolates of theB. megateriumbacteriophages (BM5 and BM10), which were isolated from Egyptian soil samples. Growth analyses indicated that the phages BM5 and BM10 have a shorter latent period (25 and 30 min, respectively) and a smaller burst size (103 and 117 PFU, respectively), in comparison to what is typical forBacillusphages. The genome sizes of the phages BM5 and BM10 were 165,031 bp and 165,213 bp, respectively, with modular organization. Bioinformatic analyses of these genomes enabled the assignment of putative functions to 97 and 65 putative ORFs, respectively. Comparative analysis of the BM5 and BM10 genome structures, in conjunction with otherB. megateriumbacteriophages, revealed relatively high levels of sequence and organizational identity. Both genomic comparisons and phylogenetic analyses support the conclusion that the sequenced phages (BM5 and BM10) belong to different sub-clusters (L5 and L7, respectively), within the L-cluster, and display different lifestyles (lysogenic and lytic, respectively). Moreover, sequenced phages encode proteins associated withBacilluspathogenesis. In addition, BM5 does not contain any tRNA sequences, whereas BM10 genome codes for 17 tRNAs.

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A20 Sample preparation for whole-genome next-generation sequencing (NGS) of hepatitis C virus (HCV) routine RNA samples

Virus Evolution ◽

10.1093/ve/vez002.019 ◽

2019 ◽

Vol 5 (Supplement_1) ◽

Author(s):

Julia Hillung ◽

María Alma Bracho ◽

Javier Pons Tamarit ◽

Fernando González-Candelas

Keyword(s):

Sample Preparation ◽

Nucleic Acids ◽

Blood Plasma ◽

Reference Genome ◽

Phylogenetic Analyses ◽

Whole Genome ◽

Library Preparation ◽

Genome Sequences ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

Abstract Next-generation sequencing (NGS) is a technique that can capture the variability of viral populations in transmission studies. The conventional sample preparation for NGS, based on amplicons, is a potential source of errors, derived from the variable affinity of specific primers for different viral variants and from irregular DNA polymerase efficiency. In this context, we propose a more reliable method for viral whole genome sample preparation, starting from nucleic acids obtained and stored with conventional procedures. Our goal was to obtain complete hepatitis C virus (HCV) genome sequences to subsequently perform extensive phylogenetic analyses. Additionally, we aimed to test the effectiveness of nuclease treatment used to remove contaminating host DNA. Nucleic acids were obtained from almost cell-free blood plasma of HCV-infected patients. As a source for Illumina library preparation, double-stranded cDNA was generated using random primers. The HCV genome was not amplified before library preparation, avoiding possible biases derived from unequal copying. To get rid of possible host contaminants in the samples, a DNase treatment step was added. Libraries were paired-end sequenced on the Illumina platform using MiSeq reagent kit v3. After conservative filtering of contaminant human reads by alignment with the human reference genome using Burrows-Wheeler Aligner (BWA), the remaining reads were mapped to the HCV reference genome using BWA. Primary maximum likelihood phylogenetic analyses were performed using ClustalW and IQTREE to infer the phylogenetic relationships of the sequenced samples in the context of complete genome sequences of the same genotype. NGS sample preparation method of HCV from blood plasma was established. Complete genome sequences of HCV could be obtained with variable coverage depending on the viral load of plasma samples. No significant reduction of host DNA proportion in DNase treated samples in comparison to the controls was observed. The new sequences clustered within the Los Alamos National Laboratory database-deposited HCV subtype 4d samples. The method can be used to obtain full-length sequences of HCV from nucleic acid samples not previously planned for NGS. No improvement was observed when DNase pre-treatment of nucleic acids extracted from blood plasma was performed.

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