scholarly journals Compartmentalized Replication of SARS-Cov-2 in Upper vs. Lower Respiratory Tract Assessed by Whole Genome Quasispecies Analysis

2020 ◽  
Vol 8 (9) ◽  
pp. 1302 ◽  
Author(s):  
Martina Rueca ◽  
Barbara Bartolini ◽  
Cesare Ernesto Maria Gruber ◽  
Antonio Piralla ◽  
Fausto Baldanti ◽  
...  
Keyword(s):  
Respiratory Tract ◽  
Lower Respiratory Tract ◽  
Bioinformatic Analysis ◽  
Whole Genome ◽  
Significant Difference ◽  
Minority Variants ◽  
Selection For ◽  
Next Generation Sequencing Ngs ◽  
Quasispecies Evolution ◽  
Generation Sequencing

We report whole-genome and intra-host variability of SARS-Cov-2 assessed by next generation sequencing (NGS) in upper (URT) and lower respiratory tract (LRT) from COVID-19 patients. The aim was to identify possible tissue-specific patterns and signatures of variant selection for each respiratory compartment. Six patients, admitted to the Intensive Care Unit, were included in the study. Thirteen URT and LRT were analyzed by NGS amplicon-based approach on Ion Torrent Platform. Bioinformatic analysis was performed using both realized in-house and supplied by ThermoFisher programs. Phylogenesis showed clade V clustering of the first patients diagnosed in Italy, and clade G for later strains. The presence of quasispecies was observed, with variants uniformly distributed along the genome and frequency of minority variants spanning from 1% to ~30%. For each patient, the patterns of variants in URT and LRT were profoundly different, indicating compartmentalized virus replication. No clear variant signature and no significant difference in nucleotide diversity between LRT and URT were observed. SARS-CoV-2 presents genetic heterogeneity and quasispecies compartmentalization in URT and LRT. Intra-patient diversity was low. The pattern of minority variants was highly heterogeneous and no specific district signature could be identified, nevertheless, analysis of samples, longitudinally collected in patients, supported quasispecies evolution.

scholarly journals COPD-Lower Respiratory Tract Infection Visual Analogue Score (c-LRTI-VAS) validation in stable and exacerbated patients with COPD

2021 ◽  
Vol 8 (1) ◽  
pp. e000761
Author(s):  
Hendrik Johannes Prins ◽  
Ruud Duijkers ◽  
Johannes M A Daniels ◽  
Thys van der Molen ◽  
Tjip S van der Werf ◽  
...  
Keyword(s):  
Tract Infection ◽  
Respiratory Tract ◽  
Respiratory Tract Infection ◽  
Lower Respiratory Tract Infection ◽  
Lower Respiratory Tract ◽  
Visual Analogue Score ◽  
Chronic Obstructive ◽  
Obstructive Pulmonary Disease ◽  
Significant Difference ◽  
Related Quality

BackgroundWe developed the chronic obstructive pulmonary disease (COPD)-Lower Respiratory Tract Infection-Visual Analogue Score (c-LRTI-VAS) in order to easily quantify symptoms during exacerbations in patients with COPD. This study aimed to validate this score.MethodsIn our study, patients with stable COPD as well as those with an acute exacerbations of COPD (AECOPD) were included. The results of c-LRTI-VAS were compared with other markers of disease activity (lung function parameters, oxygen saturation and two health related quality of life questionnaires (St Georges Respiratory Questionnaire (SGRQ) and Clinical COPD Questionnaire (CCQ)) and validity, reliability and responsiveness were assessed.ResultsEighty-eight patients with clinically stable COPD and 102 patients who had an AECOPD completed the c-LRTI-VAS questionnaire. When testing on two separate occasions for repeatability, no statistically significant difference between total scores was found 0.143 (SD 5.42) (p=0.826). Internal consistency was high across items (Cronbach’s apha 0.755). Correlation with SGRQ and CCQ total scores was moderate to high. After treatment for hospitalised AECOPD, the mean c-LRTI-VAS total score improved 8.14 points (SD 9.13; p≤0.001).Conclusionsc-LRTI-VAS showed proper validity, responsiveness to change and moderate to high correlation with other questionnaires. It, therefore, appears a reliable tool for symptom measurement during AECOPD.Trial registration numberNCT01232140.

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

Gut Pathogens ◽  
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).

scholarly journals Detection of SARS-CoV-2 and Other Infectious Agents in Lower Respiratory Tract Samples Belonging to Patients Admitted to Intensive Care Units of a Tertiary-Care Hospital, Located in an Epidemic Area, during the Italian Lockdown

2021 ◽  
Vol 9 (1) ◽  
pp. 185
Author(s):  
Adriana Calderaro ◽  
Mirko Buttrini ◽  
Sara Montecchini ◽  
Giovanna Piccolo ◽  
Monica Martinelli ◽  
...  
Keyword(s):  
Intensive Care ◽  
Respiratory Tract ◽  
Intensive Care Units ◽  
Lower Respiratory Tract ◽  
Tertiary Care Hospital ◽  
Tertiary Care ◽  
Care Hospital ◽  
Infectious Agents ◽  
Epidemic Area ◽  
Significant Difference

The aim of this study was the detection of infectious agents from lower respiratory tract (LRT) samples in order to describe their distribution in patients with severe acute respiratory failure and hospitalized in intensive care units (ICU) in an Italian tertiary-care hospital. LRT samples from 154 patients admitted to ICU from 27 February to 10 May 2020 were prospectively examined for respiratory viruses, including SARS-CoV-2, bacteria and/or fungi. SARS-CoV-2 was revealed in 90 patients (58.4%, 72 males, mean age 65 years). No significant difference was observed between SARS-CoV-2 positives and SARS-CoV-2 negatives with regard to sex, age and bacterial and/or fungal infections. Nonetheless, fungi were more frequently detected among SARS-CoV-2 positives (44/54, 81.4%, p = 0.0053). Candida albicans was the overall most frequently isolated agent, followed by Enterococcus faecalis among SARS-CoV-2 positives and Staphylococcus aureus among SARS-CoV-2 negatives. Overall mortality rate was 40.4%, accounting for 53 deaths: 37 among SARS-CoV-2 positives (mean age 69 years) and 16 among SARS-CoV-2 negatives (mean age 63 years). This study highlights the different patterns of infectious agents between the two patient categories: fungi were prevalently involved among SARS-CoV-2-positive patients and bacteria among the SARS-CoV-2-negative patients. The different therapies and the length of the ICU stay could have influenced these different patterns of infectious agents.

scholarly journals Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Elizabeth Jaworski ◽  
Rose M Langsjoen ◽  
Brooke Mitchell ◽  
Barbara Judy ◽  
Patrick Newman ◽  
...  
Keyword(s):  
Full Range ◽  
Virus Genome ◽  
Clinical Samples ◽  
Rna Recombination ◽  
Single Nucleotide ◽  
Reverse Transcription Reaction ◽  
Minority Variants ◽  
Next Generation Sequencing Ngs ◽  
Unique Molecular Identifier ◽  
Generation Sequencing

High-throughput genomics of SARS-CoV-2 is essential to characterize virus evolution and to identify adaptations that affect pathogenicity or transmission. While single-nucleotide variations (SNVs) are commonly considered as driving virus adaption, RNA recombination events that delete or insert nucleic acid sequences are also critical. Whole genome targeting sequencing of SARS-CoV-2 is typically achieved using pairs of primers to generate cDNA amplicons suitable for next-generation sequencing (NGS). However, paired-primer approaches impose constraints on where primers can be designed, how many amplicons are synthesized and requires multiple PCR reactions with non-overlapping primer pools. This imparts sensitivity to underlying SNVs and fails to resolve RNA recombination junctions that are not flanked by primer pairs. To address these limitations, we have designed an approach called ‘Tiled-ClickSeq’, which uses hundreds of tiled-primers spaced evenly along the virus genome in a single reverse-transcription reaction. The other end of the cDNA amplicon is generated by azido-nucleotides that stochastically terminate cDNA synthesis, removing the need for a paired-primer. A sequencing adaptor containing a Unique Molecular Identifier (UMI) is appended to the cDNA fragment using click-chemistry and a PCR reaction generates a final NGS library. Tiled-ClickSeq provides complete genome coverage, including the 5’UTR, at high depth and specificity to the virus on both Illumina and Nanopore NGS platforms. Here, we analyze multiple SARS-CoV-2 isolates and clinical samples to simultaneously characterize minority variants, sub-genomic mRNAs (sgmRNAs), structural variants (SVs) and D-RNAs. Tiled-ClickSeq therefore provides a convenient and robust platform for SARS-CoV-2 genomics that captures the full range of RNA species in a single, simple assay.

scholarly journals Whole genome sequencing suggests transmission of Corynebacterium diphtheriae-caused cutaneous diphtheria in two siblings, Germany, 2018

2019 ◽  
Vol 24 (2) ◽  
Author(s):  
Anja Berger ◽  
Alexandra Dangel ◽  
Tilmann Schober ◽  
Birgit Schmidbauer ◽  
Regina Konrad ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Corynebacterium Diphtheriae ◽  
Whole Genome ◽  
Next Generation ◽  
Insect Bites ◽  
Next Generation Sequencing Ngs ◽  
Ngs Data ◽  
Generation Sequencing

In September 2018, a child who had returned from Somalia to Germany presented with cutaneous diphtheria by toxigenic Corynebacterium diphtheriae biovar mitis. The child’s sibling had superinfected insect bites harbouring also toxigenic C. diphtheriae. Next generation sequencing (NGS) revealed the same strain in both patients suggesting very recent human-to-human transmission. Epidemiological and NGS data suggest that the two cutaneous diphtheria cases constitute the first outbreak by toxigenic C. diphtheriae in Germany since the 1980s.

scholarly journals Human Papillomavirus and the Development of Different Cancers

2016 ◽  
Vol 150 (3-4) ◽  
pp. 185-193 ◽  
Author(s):  
Ge Gao ◽  
David I. Smith
Keyword(s):  
Cervical Cancer ◽  
Squamous Cell Carcinomas ◽  
Human Papillomaviruses ◽  
Whole Genome ◽  
Physical Status ◽  
Cervical Cancers ◽  
Mate Pair ◽  
Almost All ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Human papillomaviruses (HPV) are responsible for the development of almost all cervical cancers. HPV is also found in 85% of anal cancer and in 50% of penile, vulvar, and vaginal cancers, and they are increasingly found in a subset of head and neck cancers, i.e., oropharyngeal squamous cell carcinomas (OPSCC). The model for how HPV causes cancer is derived from several decades of study on cervical cancer, and it is just presumed that this model is not only completely valid for cervical cancer but for all other HPV-driven cancers as well. Next-generation sequencing (NGS) has now provided the necessary tools to characterize genomic alterations in cancer cells and can precisely determine the physical status of HPV in those cells as well. We discuss recent discoveries from different applications of NGS in both cervical cancer and OPSCCs, including whole-genome sequencing and mate-pair NGS. We also discuss what NGS studies have revealed about the different ways that HPV can be involved in cancer formation, specifically comparing cervical cancer and OPSCC.

scholarly journals 2194. Procalcitonin-Guided Antibiotic Strategy for Lower Respiratory Tract Infections: Outcomes and Safety of Withholding Antibiotics

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S747-S747
Author(s):  
Don Bambino Geno Tai ◽  
Robert Goldstein
Keyword(s):  
Respiratory Tract ◽  
Hospital Mortality ◽  
Lower Respiratory Tract ◽  
Respiratory Tract Infections ◽  
Standard Of Care ◽  
Lower Respiratory Tract Infections ◽  
Case Mix Index ◽  
Significant Difference ◽  
The Mean ◽  
Tract Infections

Abstract Background Procalcitonin (PCT) is a promising and well-studied marker in differentiating viral from bacterial lower respiratory tract infections (LRTI). Antibiotics are not recommended when PCT is normal (< 0.25 μg/L). Despite this, it has not been the standard of care due to safety concerns of relying on PCT to withhold antibiotics. Methods We retrospectively reviewed all non-critical LRTI patients with normal PCT admitted in our institution from October 2018 to March 2019. They were divided into adherent group in whom antibiotics were discontinued within 24 hours and non-adherent group in whom antibiotics were continued. Cases of Legionella, Mycoplasma, and other infectious syndromes necessitating antibiotics were excluded. Complexity of cases was measured based on Centers for Medicare and Medicaid Services (CMS) case-mix index (CMI). Outcomes compared were the length of stay (LOS), in-hospital mortality, and 30-day all-cause readmissions. Results A total of 78 patients were included in the analysis, 52% (n = 41) were in adherent group and 48% (n = 37) in the non-adherent group. The mean age was 74, and majority were females (59%, n = 46). The were no significant differences between the two groups in terms of age, gender, CMI, underlying COPD/asthma, CHF (see Table 1). The adherent group had statistically shorter LOS compared with the non-adherent group after adjusting for CMI. There was no significant difference in In-hospital mortality and readmissions (see Table 2). Furthermore, the adherent group’s LOS was statistically similar to CMS average LOS (5.08 vs. 3.8, P = 0.08); compared with the non-adherent group which had statistically longer LOS compared with CMS average LOS (8.3 vs. 4.6, p Conclusion PCT is a safe tool in deciding when to withhold antibiotics on LRTI patients. It shortens LOS with no difference in mortality or readmission. Disclosures All authors: No reported disclosures.

scholarly journals Next Generation Sequencing Identifies Five Novel Mutations in Lebanese Patients with Bardet–Biedl and Usher Syndromes

Genes ◽  
2019 ◽  
Vol 10 (12) ◽  
pp. 1047 ◽  
Author(s):  
Lama Jaffal ◽  
Wissam H Joumaa ◽  
Alexandre Assi ◽  
Charles Helou ◽  
George Cherfan ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Usher Syndrome ◽  
Bioinformatic Analysis ◽  
Next Generation ◽  
Novel Mutations ◽  
Pathogenic Variants ◽  
Whole Exome ◽  
Targeted Ngs ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Aim: To identify disease-causing mutations in four Lebanese families: three families with Bardet–Biedl and one family with Usher syndrome (BBS and USH respectively), using next generation sequencing (NGS). Methods: We applied targeted NGS in two families and whole exome sequencing (WES) in two other families. Pathogenicity of candidate mutations was evaluated according to frequency, conservation, in silico prediction tools, segregation with disease, and compatibility with inheritance pattern. The presence of pathogenic variants was confirmed via Sanger sequencing followed by segregation analysis. Results: Most likely disease-causing mutations were identified in all included patients. In BBS patients, we found (M1): c.2258A > T, p. (Glu753Val) in BBS9, (M2): c.68T > C; p. (Leu23Pro) in ARL6, (M3): c.265_266delTT; p. (Leu89Valfs*11) and (M4): c.880T > G; p. (Tyr294Asp) in BBS12. A previously known variant (M5): c.551A > G; p. (Asp184Ser) was also detected in BBS5. In the USH patient, we found (M6): c.188A > C, p. (Tyr63Ser) in CLRN1. M2, M3, M4, and M6 were novel. All of the candidate mutations were shown to be likely disease-causing through our bioinformatic analysis. They also segregated with the corresponding phenotype in available family members. Conclusion: This study expanded the mutational spectrum and showed the genetic diversity of BBS and USH. It also spotlighted the efficiency of NGS techniques in revealing mutations underlying clinically and genetically heterogeneous disorders.

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