We developed a metagenomic next-generation sequencing (mNGS) test using cell-free DNA from body fluids to identify pathogens. The performance of mNGS testing of 182 body fluids from 160 patients with ...acute illness was evaluated using two sequencing platforms in comparison to microbiological testing using culture, 16S bacterial PCR and/or 28S-internal transcribed ribosomal gene spacer (28S-ITS) fungal PCR. Test sensitivity and specificity of detection were 79 and 91% for bacteria and 91 and 89% for fungi, respectively, by Illumina sequencing; and 75 and 81% for bacteria and 91 and 100% for fungi, respectively, by nanopore sequencing. In a case series of 12 patients with culture/PCR-negative body fluids but for whom an infectious diagnosis was ultimately established, seven (58%) were mNGS positive. Real-time computational analysis enabled pathogen identification by nanopore sequencing in a median 50-min sequencing and 6-h sample-to-answer time. Rapid mNGS testing is a promising tool for diagnosis of unknown infections from body fluids.
An outbreak of betacoronavirus severe acute respiratory syndrome (SARS)-CoV-2 began in Wuhan, China in December 2019. COVID-19, the disease associated with SARS-CoV-2 infection, rapidly spread to ...produce a global pandemic. We report development of a rapid (<40 min), easy-to-implement and accurate CRISPR-Cas12-based lateral flow assay for detection of SARS-CoV-2 from respiratory swab RNA extracts. We validated our method using contrived reference samples and clinical samples from patients in the United States, including 36 patients with COVID-19 infection and 42 patients with other viral respiratory infections. Our CRISPR-based DETECTR assay provides a visual and faster alternative to the US Centers for Disease Control and Prevention SARS-CoV-2 real-time RT-PCR assay, with 95% positive predictive agreement and 100% negative predictive agreement.
Background Antimicrobial resistance (AMR) is rising at an alarming rate and complicating the management of infectious diseases including lower respiratory tract infections (LRTI). Metagenomic ...next-generation sequencing (mNGS) is a recently established method for culture-independent LRTI diagnosis, but its utility for predicting AMR has remained unclear. We aimed to assess the performance of mNGS for AMR prediction in bacterial LRTI and demonstrate proof of concept for epidemiological AMR surveillance and rapid AMR gene detection using Cas9 enrichment and nanopore sequencing. Methods We studied 88 patients with acute respiratory failure between 07/2013 and 9/2018, enrolled through a previous observational study of LRTI. Inclusion criteria were age greater than or equai to 18, need for mechanical ventilation, and respiratory specimen collection within 72 h of intubation. Exclusion criteria were decline of study participation, unclear LRTI status, or no matched RNA and DNA mNGS data from a respiratory specimen. Patients with LRTI were identified by clinical adjudication. mNGS was performed on lower respiratory tract specimens. The primary outcome was mNGS performance for predicting phenotypic antimicrobial susceptibility and was assessed in patients with LRTI from culture-confirmed bacterial pathogens with clinical antimicrobial susceptibility testing (n = 27 patients, n = 32 pathogens). Secondary outcomes included the association between hospital exposure and AMR gene burden in the respiratory microbiome (n = 88 patients), and AMR gene detection using Cas9 targeted enrichment and nanopore sequencing (n = 10 patients). Results Compared to clinical antimicrobial susceptibility testing, the performance of respiratory mNGS for predicting AMR varied by pathogen, antimicrobial, and nucleic acid type sequenced. For gram-positive bacteria, a combination of RNA + DNA mNGS achieved a sensitivity of 70% (95% confidence interval (CI) 47-87%) and specificity of 95% (CI 85-99%). For gram-negative bacteria, sensitivity was 100% (CI 87-100%) and specificity 64% (CI 48-78%). Patients with hospital-onset LRTI had a greater AMR gene burden in their respiratory microbiome versus those with community-onset LRTI (p = 0.00030), or those without LRTI (p = 0.0024). We found that Cas9 targeted sequencing could enrich for low abundance AMR genes by > 2500-fold and enabled their rapid detection using a nanopore platform. Conclusions mNGS has utility for the detection and surveillance of resistant bacterial LRTI pathogens.
Metagenomic next-generation sequencing (mNGS), the shotgun sequencing of RNA and DNA from clinical samples, has proved useful for broad-spectrum pathogen detection and the genomic surveillance of ...viral outbreaks. An additional target enrichment step is generally needed for high-sensitivity pathogen identification in low-titre infections, yet available methods using PCR or capture probes can be limited by high cost, narrow scope of detection, lengthy protocols and/or cross-contamination. Here, we developed metagenomic sequencing with spiked primer enrichment (MSSPE), a method for enriching targeted RNA viral sequences while simultaneously retaining metagenomic sensitivity for other pathogens. We evaluated MSSPE for 14 different viruses, yielding a median tenfold enrichment and mean 47% (±16%) increase in the breadth of genome coverage over mNGS alone. Virus detection using MSSPE arboviral or haemorrhagic fever viral panels was comparable in sensitivity to specific PCR, demonstrating 95% accuracy for the detection of Zika, Ebola, dengue, chikungunya and yellow fever viruses in plasma samples from infected patients. Notably, sequences from re-emerging and/or co-infecting viruses that have not been specifically targeted a priori, including Powassan and Usutu, were successfully enriched using MSSPE. MSSPE is simple, low cost, fast and deployable on either benchtop or portable nanopore sequencers, making this method directly applicable for diagnostic laboratory and field use.
Mucosal-associated Escherichia coli are commonly found in inflamed tissues during inflammatory bowel disease (IBD). These bacteria often possess an adherent and invasive phenotype but lack ...virulence-associated features of well-described intestinal E. coli pathogens, and are of diverse serology and phylotypes, making it difficult to correlate strain characteristics with exacerbations of disease.
The genome sequences of 14 phenotypically assigned adherent-invasive Escherichia coli (AIEC) isolates obtained from intestinal biopsies of patients with IBD were compared with the genome sequences of 37 other pathogenic and commensal E. coli available from public databases.
Core genome-based phylogenetic analyses and genome-wide comparison of genetic content established the existence of a closely related cluster of AIEC strains with 3 distinct genetic insertions differentiating them from commensal E. coli. These strains are of the B2 phylotype have a variant type VI secretion system (T6SS-1), and are highly related to extraintestinal pathogenic E. coli, suggesting that these 2 clinically distinct pathovars have common virulence strategies. Four other mucosally adherent E. coli strains from patients with IBD were of diverse phylogenetic origins and lacked the 3 genetic features, suggesting that they are not related to the B2 AIEC cluster. Although AIEC are often considered as having a unique association with Crohn's disease, isolates from Crohn's disease and ulcerative colitis were genetically indistinguishable.
B2 AIEC thus represent a closely related cluster of IBD-associated E. coli strains that are distinct from normal commensal isolates, and which should be considered separately from the phenotypically similar but genetically distinct non-B2 AIEC strains when considering their association with intestinal pathogenesis.
Background. In 2010, Winnipeg, Canada, experienced a doubling of invasive pneumococcal disease (IPD) rates, with a significant increase in the number of cases due to Streptococcus pneumoniae serotype ...12F, which previously had accounted for very few cases each year. Methods. All serotype 12F IPD cases reported between September 2009 and January 2011 were reviewed. Pulsed-field gel electrophoresis (PFGE) and multilocus variable number tandem repeat analysis (MLVA) were conducted on all isolates. PFGE and MLVA patterns identified several possible clusters. Additional interviews were conducted to obtain information on risk factors and outcomes. Results. Between September 2009 and January 2011, 169 cases of IPD were identified. The number of IPD cases due to 12F serotype increased sharply from about 3–4 cases per year (6% of IPD cases) in 2007–2009 to 28 (29%) in 2010. All 12F isolates belonged to a single sequence type (ST218), and they were generally susceptible to penicillin and fluoroquinolones but not to erythromycin. Compared with cases caused by other serotypes, patients with serotype 12F were more likely to be homeless, reside in low-income inner-city communities, and engage in substance abuse, including intravenous and crack cocaine use. Subclusters identified using MLVA had even higher rates of homelessness and substance use. Conclusions. An immunization campaign targeting high-risk groups was undertaken with pneumococcal polysaccharide vaccine, and subsequently rates of serotype 12F decreased. To our knowledge, this is the largest documented community outbreak of serotype 12F IPD and the first report of an outbreak of IPD serotype 12F in a marginalized urban population in Canada.
Associations between vaccine breakthrough cases and infection by different SARS coronavirus 2 (SARS-CoV-2) variants have remained largely unexplored. Here we analysed SARS-CoV-2 whole-genome ...sequences and viral loads from 1,373 persons with COVID-19 from the San Francisco Bay Area from 1 February to 30 June 2021, of which 125 (9.1%) were vaccine breakthrough infections. Vaccine breakthrough infections were more commonly associated with circulating antibody-resistant variants carrying ≥1 mutation associated with decreased antibody neutralization (L452R/Q, E484K/Q and/or F490S) than infections in unvaccinated individuals (78% versus 48%, P = 1.96 × 10
). Differences in viral loads were non-significant between unvaccinated and fully vaccinated cases overall (P = 0.99) and according to lineage (P = 0.09-0.78). Symptomatic vaccine breakthrough infections had comparable viral loads (P = 0.64), whereas asymptomatic breakthrough infections had decreased viral loads (P = 0.023) compared with infections in unvaccinated individuals. In 5 cases with serial samples available for serologic analyses, vaccine breakthrough infections were found to be associated with low or undetectable neutralizing antibody levels attributable to an immunocompromised state or infection by an antibody-resistant lineage. Taken together, our results show that vaccine breakthrough infections are overrepresented by antibody-resistant SARS-CoV-2 variants, and that symptomatic breakthrough infections may be as efficient in spreading COVID-19 as unvaccinated infections, regardless of the infecting lineage.
The introduction of pneumococcal conjugate vaccines has led to the emergence of non-vaccine serotypes, which contributed to invasive pneumococcal disease in Canada and worldwide. A significant ...increase in the prevalence of non-13-valent pneumococcal conjugate vaccine (PCV-13)-included serotypes 22F, 15A, and 8 was observed from 2009 to 2013 in Ontario (all p values<0.01). In this study, whole genome sequencing was conducted on the 25 isolates of serotype 22F, seven of 15A and 10 of 8 to investigate the population structure and antibiotic resistance. All seven serotype 15A isolates were found to be multidrug resistant. From whole genome analysis, we observed recombination events among serotypes 22F, 15A and 8 populations. Serotype 22F (ST433) has emerged into two sub-populations, with 28% (7/25) exhibiting recombination events, and five also acquiring macrolide resistance as a result of recombination. This study enhances the knowledge on the molecular evolution of emerging non-PCV-13 vaccine serotype 22F, including acquisition of resistance genes through recombination events. It underpins the importance of whole genome sequencing in studying Streptococcus pneumoniae population structures and dynamics, and its utility in molecular surveillance.
•Identified the emergence of non-PCV-13 serotypes associated with IPD in the adults, Canada.•Population structure and antimicrobial susceptibility profiles of 22F, 15A and 8 serotypes studied.•The 22F serotype, the most predominant serotype in 2013, has diversified into two sub-clusters and acquired macrolide resistance.•We observed the link between recombination and acquisition of antimicrobial resistance genes•Study highlights role of whole genome sequencing in surveillance in the public health laboratory
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