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Hemocyanins are multimeric oxygen transport proteins present in the blood of arthropods and molluscs, containing up to 8 oxygen-binding functional units per monomer. In molluscs, hemocyanins are assembled in decamer 'building blocks' formed of 5 dimer 'plates', routinely forming didecamer or higher-order assemblies with d5 or c5 symmetry. Here we describe the cryoEM structures of the didecamer (20-mer) and tridecamer (30-mer) forms of a novel hemocyanin from the slipper limpet Crepidula fornicata (SLH) at 7.0 and 4.7 Å resolution respectively. We show that two decamers assemble in a 'tail-tail' configuration, forming a partially capped cylinder, with an additional decamer adding on in 'head-tail' configuration to make the tridecamer. Analysis of SLH samples shows substantial heterogeneity, suggesting the presence of many higher-order multimers including tetra- and pentadecamers, formed by successive addition of decamers in head-tail configuration. Retrieval of sequence data for a full-length isoform of SLH enabled the use of Alphafold to produce a molecular model of SLH, which indicated the formation of dimer slabs with high similarity to those found in keyhole limpet hemocyanin. The fit of the molecular model to the cryoEM density was excellent, showing an overall structure where the final two functional units of the subunit (FU-g and FU-h) form the partial cap at one end of the decamer, and permitting analysis of the subunit interfaces governing the assembly of tail-tail and head-tail decamer interactions as well as potential sites for N-glycosylation. Our work contributes to the understanding of higher-order oligomer formation in molluscan hemocyanins and demonstrates the utility of Alphafold for building accurate structural models of large oligomeric proteins. [ABSTRACT FROM AUTHOR]

Treatment of rifampin-monoresistant/multidrug-resistant Tuberculosis (RR/MDR-TB) requires long treatment courses, complicated by frequent adverse events and low success rates. Incidence of RR/MDR-TB in Canada is low and treatment practices are variable due to the infrequent experience and challenges with drug access. We undertook a retrospective cohort study of all RR/MDR-TB cases in Alberta, Canada from 2007–2017 to explore the epidemiology and outcomes in our low incidence setting. We performed a descriptive analysis of the epidemiology, treatment regimens and associated outcomes, calculating differences in continuous and discrete variables using Student's t and Chi-squared tests, respectively. We identified 24 patients with RR/MDR-TB. All patients were foreign-born with the median time to presentation after immigration being 3 years. Prior treatment was reported in 46%. Treatment was individualized. All patients achieved sputum culture conversion within two months of treatment initiation. The median treatment duration after culture conversion was 18 months (IQR: 15–19). The mean number of drugs utilized during the intensive phase was 4.3 (SD: 0.8) and during the continuation phase was 3.3 (SD: 0.9) and the mean adherence to medications was 95%. Six patients completed national guideline-concordant therapy, with many patients developing adverse events (79%). Treatment success (defined as completion of prescribed therapy or cure) was achieved in 23/24 patients and no acquired drug resistance or relapse was detected over 1.8 years of median follow-up. Many cases were captured upon immigration assessment, representing important prevention of community spread. Despite high rates of adverse events and short treatment compared to international guidelines, success in our cohort was very high at 96%. This is likely due to individualization of therapy, frequent use of medications with high effectiveness, intensive treatment support, and early sputum conversion seen in our cohort. There should be ongoing exploration of treatment shortening with well-tolerated, efficacious oral agents to help patients achieve treatment completion. [ABSTRACT FROM AUTHOR]

Isoniazid resistant Mycobacterium tuberculosis (Hr-TB) is the most frequently encountered TB resistance phenotype in North America but limited data exist on the effectiveness of current therapeutic regimens. Ineffective treatment of Hr-TB increases patient relapse and anti-mycobacterial resistance, specifically MDR-TB. We undertook a multi-centre, retrospective review of culture-positive Hr-TB patients in Alberta, Canada (2007–2017). We assessed incidence and treatment outcomes, with a focus on fluoroquinolone (FQ)-containing regimens, to understand the risk of unsuccessful outcomes. Rates of Hr-TB were determined using the mid-year provincial population and odds of unsuccessful treatment was calculated using a Fisher's Exact test. One hundred eight patients of median age 37 years (IQR: 26–50) were identified with Hr-TB (6.3%), 98 of whom were able to be analyzed. Seven percent reported prior treatment. Rate of foreign birth was high (95%), but continent of origin did not predict Hr-TB (p = 0.47). Mean compliance was 95% with no difference between FQ and non-FQ regimens (p = 1.00). Treatment success was high (91.8%). FQ-containing regimens were frequently initiated (70%), with no difference in unsuccessful outcomes compared to non-FQ-containing regimens (5.8% vs. 13.8%, OR 0.4, 95% CI 0.1–2.3, p = 0.23). Only one patient (1%) utilizing a less common non-FQ-based regimen including two months of pyrazinamide developed secondary multidrug resistance. Unsuccessful treatment was low (<10%) relative to comparable literature (~15%) and showed similar outcomes for FQ and non-FQ-based regimens and no deficit to those using intermittent fluoroquinolones in the continuation phase of treatment. Our findings are similar to recent data, however prospective, randomized trials of adequate power are needed to determine the optimal treatment for Hr-TB. [ABSTRACT FROM AUTHOR]

Introduction: Compliance with the recommendation that all tuberculosis (TB) patients be tested for human immunodeficiency virus (HIV) has not yet been achieved in Canada or globally. Methods: The experience of “opt-out” HIV testing of TB patients in the Province of Alberta, Canada is described over a 10-year period, 2003–2012. Testing rates are reported before and after the introduction of the “opt-out” approach. Risk factors for HIV seropositivity are described and demographic, clinical and laboratory characteristics of TB patients who were newly diagnosed versus previously diagnosed with HIV are compared. Genotypic clusters, defined as groups of two or more cases whose isolates of Mycobacterium tuberculosis had identical DNA fingerprints over the 10-year period or within 2 years of one another, were analyzed for their ability to predict HIV co-infection. Results: HIV testing rates were 26% before and 90% after the introduction of “opt-out” testing. During the “opt-out” testing years those <15 or >64 years of age at diagnosis were less likely to have been tested. In those tested the prevalence of HIV was 5.6%. In the age group 15–64 years, risk factors for HIV were: age (35–64 years), Canadian-born Aboriginal or foreign-born sub-Saharan African origin, and combined respiratory and non-respiratory disease. Compared to TB patients previously known to be HIV positive, TB patients newly discovered to be HIV positive had more advanced HIV disease (lower CD4 counts; higher viral loads) at diagnosis. Large cluster size was associated with Aboriginal ancestry. Cluster size predicted HIV co-infection in Aboriginal peoples when clusters included all cases reported over 10 years but not when clusters included cases reported within 2 years of one another. Conclusion: “Opt-out” HIV testing of TB patients is effective and well received. Universal HIV testing of TB patients (>80% of patients tested) has immediate (patients) and longer-term (TB/HIV program planning) benefits. [ABSTRACT FROM AUTHOR]