No abstract

Objectives The COVID-19 pandemic and ensuing public health emergency has emphasized the need to study SARS-CoV-2 pathogenesis. The human microbiome has been shown to regulate the host immune system and may influence host susceptibility to viral infection, as well as disease severity. Several studies have assessed whether compositional alterations in the nasopharyngeal microbiota are associated with SARS-CoV-2 infection. However, the results of these studies were varied, and many did not account for disease severity. This study aims to examine whether compositional differences in the nasopharyngeal microbiota are associated with SARS-CoV-2 infection status and disease severity. Methods We performed Nanopore full-length 16S rRNA sequencing on 194 nasopharyngeal swab specimens from hospitalized and community-dwelling SARS-CoV-2-infected and uninfected individuals. Sequence data analysis was performed using the BugSeq 16S analysis pipeline. Results We found significant beta (PERMANOVA p < 0.05), but not alpha (Kruskal-Wallis p > 0.05) diversity differences in the nasopharyngeal microbiota among our study groups. We identified several differentially abundant taxa associated with SARS-CoV-2 infection status and disease severity using ALDEx2. Finally, we observed a trend towards higher abundance of Enterobacteriaceae in specimens from hospitalized SARS-CoV-2-infected patients. Conclusions This study identified several alterations in the nasopharyngeal microbiome associated with SARS-CoV-2 infection status and disease severity. Understanding the role of the microbiome in infection susceptibility and severity may open new avenues of research for disease prevention and treatment.

Objectives The COVID-19 pandemic has underscored the need for rapid novel diagnostic strategies. Metagenomic Next-Generation Sequencing (mNGS) may allow for the detection of pathogens that can be missed in targeted assays. The goal of this study was to assess the performance of nanopore-based Sequence-Independent Single Primer Amplification (SISPA) for the detection and characterization of SARS-CoV-2. Methods We performed mNGS on clinical samples and designed a diagnostic classifier that corrects for barcode crosstalk between specimens. Phylogenetic analysis was performed on genome assemblies. Results Our assay yielded 100% specificity overall and 95.2% sensitivity for specimens with a RT-PCR cycle threshold value less than 30. We assembled 10 complete, and one near-complete genomes from 20 specimens that were classified as positive by mNGS. Phylogenetic analysis revealed that 10/11 specimens from British Columbia had a closest relative to another British Columbian specimen. We found 100% concordance between phylogenetic lineage assignment and Variant of Concern (VOC) PCR results. Our assay was able to distinguish between the Alpha and Gamma variants, which was not possible with the current standard VOC PCR being used in British Columbia. Conclusions This study supports future work examining the broader feasibility of nanopore mNGS as a diagnostic strategy for the detection and characterization of viral pathogens.

The BioFire FilmArray Respiratory Panel (FA RP) is a rapid multiplexed molecular assay approved for detection of viral and atypical bacterial pathogens in nasopharyngeal specimens. This study aimed to evaluate the performance of the BioFire FilmArray Respiratory Panel v1.7 on bronchoscopy specimens. We tested 133 bronchial specimens (87 archived and 46 prospectively collected) with the FA RP and compared the results to the Luminex NxTAG Respiratory Pathogen Panel (NxTAG RPP). After discordant analysis, 123 specimens gave concordant results using the FA RP and the NxTAG RPP for an overall agreement of 93.9% (kappa = 0.88 [95% CI 0.80-0.96]), a positive percent agreement of 93.7% (95% CI 83.7-97.7) and a negative percent agreement of 94.1% (95% CI 84.9-98.1). In conclusion, the BioFire FilmArray RP performed reliably to detect a broad range of respiratory pathogens in bronchoscopy specimens.

A large gap remains between sequencing a microbial community and characterizing all of the organisms inside of it. Here we develop a novel method to taxonomically bin metagenomic assemblies through alignment of contigs against a reference database. We show that this workflow, BugSplit, bins metagenome-assembled contigs to species with a 33% absolute improvement in F1-score when compared to alternative tools. We perform nanopore mNGS on patients with COVID-19, and using a reference database predating COVID-19, demonstrate that BugSplit’s taxonomic binning enables sensitive and specific detection of a novel coronavirus not possible with other approaches. When applied to nanopore mNGS data from cases of Klebsiella pneumoniae and Neisseria gonorrhoeae infection, BugSplit’s taxonomic binning accurately separates pathogen sequences from those of the host and microbiota, and unlocks the possibility of sequence typing, in silico serotyping, and antimicrobial resistance prediction of each organism within a sample. BugSplit is available at https://bugseq.com/academic.

In light of the present pandemic of novel coronavirus disease 2019 (COVID-19) and the unprecedented high demand for SARS-CoV-2 testing worldwide, there are shortages of established specimen collection devices for respiratory viral testing for diagnostic microbiology laboratories. This creates a necessity to validate unverified collection devices from manufacturers that may not be a registered supplier for medical devices. As clinical laboratories do not routinely perform quality control of established collection devices, there is a need to have a systematic, robust approach towards the assessment of substitute unregistered collection swabs and viral transport media (VTM). A discussion of the aspects requiring consideration when determining the suitability and implementation of new collection devices is presented. These specific assessment criteria include an inspection of device integrity, determination of swab and VTM sterility and in vitro performance, VTM stability and examination of clinical performance of the device. This method was used in a front-line medical microbiology laboratory on swabs and VTM from an unregistered manufacturer, with suboptimal results which precluded implementation. As the pandemic continues, it will be important for diagnostic laboratories to adopt a flexible and streamlined approach towards maintaining adequate supply chain for testing reagents and materials.

No abstract

The COVID-19 pandemic has underscored the need for rapid novel diagnostic strategies to detect and characterize pathogens from clinical specimens. The MinION sequencing device allows for rapid, cost-effective, high-throughput sequencing; useful features for translation to clinical laboratory settings. Metagenomic Next-Generation Sequencing (mNGS) approaches provide the opportunity to examine the entire genomic material of a sample; allowing for detection of emerging and clinically relevant pathogens that may be missed in targeted assays. Here we present a pilot study on the performance of Sequence-Independent Single Primer Amplification (SISPA) to amplify RNA randomly for the detection and characterization of SARS-CoV-2. We designed a classifier that corrects for barcode crosstalk between specimens. Our assay yielded 100% specificity overall and 95.2% sensitivity for specimens with a RT-qPCR cycle threshold value less than 30. We assembled 10 complete (>95% coverage at 20x depth), and one near-complete (>80% coverage at 20x depth) genomes from 20 specimens that were classified as positive by mNGS. We characterized these genomes through phylogenetic analysis and found that 10/11 specimens from British Columbia had a closest relative to another British Columbian specimen. Of five samples that we had both assembled genomes, as well as Variant of Concern (VOC) PCR results, we found 100% concordance between these results. Additionally, our assay was able to distinguish between the Alpha and Gamma variants, which was not possible with our VOC PCR technique. This study supports future work examining the broader feasibility of SISPA as a diagnostic strategy for the detection and characterization of viral pathogens.