Targeted amplification-based whole genome sequencing of Monkeypox virus in clinical specimens.

IMPORTANCE: We present a protocol to efficiently sequence genomes of the MPXV-causing mpox. This enables researchers and public health agencies to acquire high-quality genomic data using a rapid and cost-effective approach. Genomic data can be used to conduct surveillance and investigate mpox outbreaks. We present 91 mpox genomes that show the diversity of the 2022 mpox outbreak in Ontario, Canada.

Evaluating the use of whole genome sequencing for the investigation of a large mumps outbreak in Ontario, Canada.

In 2017 Ontario experienced the largest mumps outbreak in the province in 8 years, at a time when multiple outbreaks were occurring across North America. Of 259 reported cases, 143 occurred in Toronto, primarily among young adults. Routine genotyping of the small hydrophobic gene indicated that the outbreak was due to mumps virus genotype G. We performed a retrospective study of whole genome sequencing of 26 mumps virus isolates from early in the outbreak, using a tiling amplicon method. Results indicated that two of the cases were genetically divergent, with the remaining 24 cases belonging to two major clades and one minor clade. Phylogeographic analysis confirmed circulation of virus from each clade between Toronto and other regions in Ontario. Comparison with other genotype G strains from North America suggested that the presence of co-circulating major clades may have been due to separate importation events from outbreaks in the United States. A transmission network analysis performed with the software program TransPhylo was compared with previously collected epidemiological data. The transmission tree correlated with known epidemiological links between nine patients and identified new potential clusters with no known epidemiological links.

Structural cortical network reorganization associated with early conversion to multiple sclerosis.

Brain structural covariance networks (SCNs) based on pairwise statistical associations of cortical thickness data across brain areas reflect underlying physical and functional connections between them. SCNs capture the complexity of human brain cortex structure and are disrupted in neurodegenerative conditions. However, the longitudinal assessment of SCN dynamics has not yet been explored, despite its potential to unveil mechanisms underlying neurodegeneration. Here, we evaluated the changes of SCNs over 12 months in patients with a first inflammatory-demyelinating attack of the Central Nervous System and assessed their clinical relevance by comparing SCN dynamics of patients with and without conversion to multiple sclerosis (MS) over one year. All subjects underwent clinical and brain MRI assessments over one year. Brain cortical thicknesses for each subject and time point were used to obtain group-level between-area correlation matrices from which nodal connectivity metrics were obtained. Robust bootstrap-based statistical approaches (allowing sampling with replacement) assessed the significance of longitudinal changes. Patients who converted to MS exhibited significantly greater network connectivity at baseline than non-converters (p = 0.02) and a subsequent connectivity loss over time (p = 0.001-0.02), not observed in non-converters' network. These findings suggest SCN analysis is sensitive to brain tissue changes in early MS, reflecting clinically relevant aspects of the condition. However, this is preliminary work, indicated by the low sample sizes, and its results and conclusions should be treated with caution and confirmed with larger cohorts.

Vancomycin-Variable Enterococcal Bacteremia.

Vancomycin-variable enterococcus (VVE) is an emerging pathogen. VVE isolates initially appear phenotypically susceptible to vancomycin but possesses the vanA gene and can develop in vitro and in vivo resistance to vancomycin. We report a case of VVE bacteremia and describe how VVE poses diagnostic and therapeutic dilemmas.

Interim estimates of 2013/14 vaccine effectiveness against influenza A(H1N1)pdm09 from Canada s sentinel surveillance network, January 2014.

The 2013/14 influenza season to date in Canada has been characterised by predominant (90%) A(H1N1)pdm09 activity. Vaccine effectiveness (VE) was assessed in January 2014 by Canada's sentinel surveillance network using a test-negative case-control design. Interim adjusted-VE against medically-attended laboratory-confirmed influenza A(H1N1)pdm09 infection was 74% (95% CI: 58-83). Relative to vaccine, A(H1N1)pdm09 viruses were antigenically similar and genetically well conserved, with most showing just three mutations across the 50 amino acids comprising antigenic sites of the haemagglutinin protein.

Variant influenza A (H1N1) virus infection in Canada.

There has been an increase in influenza A variant detections in the US in recent years. In September 2012, an Ontario resident was diagnosed with influenza A (H1N1) variant infection. The demonstrated cross reactivity with the A(H1N1)pdm09 H1 gene CDC realtime PCR suggests that laboratories that only use the pdm09 H1 gene PCR to confirm this subtype would incorrectly report this variant as a A(H1N1)pdm09 subtype unless they were doing further molecular investigations.

Interim estimates of influenza vaccine effectiveness in 2012/13 from Canada's sentinel surveillance network, January 2013.

The 2012/13 influenza season in Canada has been characterised to date by early and moderately severe activity, dominated (90%) by the A(H3N2) subtype. Vaccine effectiveness (VE) was assessed in January 2013 by Canada's sentinel surveillance network using a test-negative case-control design. Interim adjusted-VE against medically attended laboratory-confirmed influenza A(H3N2) infection was 45% (95% CI: 13-66). Influenza A(H3N2) viruses in Canada are similar to the vaccine, based on haemagglutination inhibition; however, antigenic site mutations are described in the haemagglutinin gene.

Progressive multifocal leukoencephalopathy: a review of the neuroimaging features and differential diagnosis.

Progressive multifocal leukoencephalopathy (PML) is an uncommon and often fatal demyelinating disease of human central nervous system, which is caused by reactivation of the polyomavirus JC (JCV). PML generally occurs in patients with profound immunosuppression such as AIDS patients. Recently, a number of PML cases have been associated with administration of natalizumab for treatment of multiple sclerosis (MS) patients. Diagnosis and management of PML became a major concern after its occurrence in multiple sclerosis patients treated with natalizumab. Diagnosis of PML usually rests on neuroimaging in the appropriate clinical context and is further confirmed by cerebrospinal fluid polymerase chain reaction (PCR) for JCV DNA. Treatment with antiretroviral therapies in HIV-seropositive patients or discontinuing natalizumab in MS patients with PML may lead to the development of immune reconstitution inflammatory syndrome (IRIS) which presents with deterioration of the previous symptoms and may lead to death. In patients under treatment with monoclonal antibodies in routine practice, or new ones in ongoing clinical trials, differentiating PML from new MS lesions on brain MRI is critical for both the neurologists and neuroradiologists. In this review, we discuss the clinical features, neuroimaging manifestations of PML, IRIS and neuroimaging clues to differentiate new MS lesions from PML. In addition, various neuroimaging features of PML on the non-conventional MR techniques such as diffusion-weighted imaging (DWI), diffusion tensor imaging (DTI), and MR spectroscopy (MRS) are discussed.

Development of a novel real-time reverse-transcriptase PCR method for the detection of H275Y positive influenza A H1N1 isolates.

During the 2007-2008 influenza season global strain surveillance for antiviral resistance revealed the sudden emergence of oseltamivir resistance in influenza A H1N1 isolates. Although oseltamivir resistance rates vary from region to region, 16% of isolates tested globally were found to be oseltamivir resistant by a histidine to tyrosine mutation of residue 275 of the neuraminidase gene of influenza A. In order to implement effective resistance testing locally a novel real-time reverse-transcriptase PCR (RT-PCR) assay was developed for the detection of the H275Y mutation. To evaluate this method, 40 oseltamivir resistant and 61 oseltamivir sensitive H1N1 influenza isolates were tested using Sanger sequencing, which is the reference method for detection of resistance, pyrosequencing and the novel H275Y RT-PCR assay. In comparison to Sanger sequencing, the sensitivity and specificity of the H275Y RT-PCR assay were 100% (40/40) and 100% (61/61) respectively, while the sensitivity and specificity of pyrosequencing were 100% (40/40) and 97.5% (60/61) respectively. Although all three methods were effective in detecting the H275Y mutation associated with oseltamivir resistance, the H275Y RT-PCR assay was the most rapid and could easily be incorporated into an influenza subtyping protocol.

Genetic microheterogeneity of emerging H275Y influenza virus A (H1N1) in Toronto, Ontario, Canada from the 2007-2008 respiratory season.

BACKGROUND: The H275Y mutation (H274Y in N2 numbering) in the neuraminidase (NA) gene (segment 6) of the influenza virus A (H1N1) genome is linked to oseltamivir resistance. OBJECTIVES: To determine the percentage of influenza virus A (H1N1) isolates that carry the H275Y mutation in the NA gene in Toronto, Ontario, Canada and to characterize select oseltamivir resistant and susceptible isolates using sequence analysis. STUDY DESIGN: Sanger sequencing was used to determine strain type and H275Y mutations based on partial sequencing of the hemagglutinin (HA) (segment 4) and NA genes. Mutations in the NS1 gene (segment 8) were determined by Sanger sequencing and pyrosequencing. Statistical analysis of demographics and proportions of H275 and H275Y isolates with mutations was carried out using chi(2) analyses. RESULTS: The HA gene of influenza virus A (H1N1) isolates collected during the 2007-2008 respiratory season was most like influenza A/Brisbane/59/2007, Clade 2, subclade B. Seventeen percent of these isolates possessed the H275Y NA mutation associated with oseltamivir resistance. H275Y isolates were more likely than H275 isolates to have the mutations A209T and R224G in NS1 (chi(2)=284.9, df=2, p<0.0001). CONCLUSIONS: During the 2007-2008 influenza season in Toronto, Ontario, Canada, 17% of influenza virus A (H1N1) isolates carried the H275Y mutation associated with oseltamivir resistance. These H275Y isolates were more likely than H275 isolates to exhibit unique microheterogeneity in the gene encoding the NS1 protein.

Differential patterns of amantadine-resistance in influenza A (H3N2) and (H1N1) isolates in Toronto, Canada.

BACKGROUND: Molecular methods were used to characterize influenza A (H1N1) and (H3N2) strains and to identify amantadine-resistance. OBJECTIVES: To compare proportions of amantadine-resistant influenza A (H1N1) and (H3N2) isolates in the Greater Toronto Area. STUDY DESIGN: Isolates of influenza A (H1N1) and (H3N2) were strain typed using molecular methods. Pyrosequencing for point mutations in the transmembrane domain of the M2 proton channel was undertaken. Proportions of amantadine-resistant and susceptible isolates were compared using the The Fisher's exact test. RESULTS: 96% of the 49 influenza A (H3N2) isolates and none of the influenza A (H1N1) tested carried a point mutation in the M gene coding for the M2 protein. Influenza A (H3N2) isolates were more likely to carry an amantadine-resistance associated mutation than influenza A (H1N1) isolates (Fishers's exact test, P<0.0001). CONCLUSIONS: : Characterization of amantadine-resistance in influenza A (H1N1) isolates should utilize a variety of different methods including sub-typing, strain typing, and direct sequencing for point mutations associated with amantadine-resistance.