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The WorldWide Antimalarial Resistance Network (WWARN) has been shortlisted for a 2017 Times Higher Education Award in the International Collaboration of the Year category.
Impact of a package of diagnostic tools, clinical algorithm, and training and communication on outpatient acute fever case management in low- and middle-income countries: protocol for a randomized controlled trial.
BackgroundThe management of acute febrile illnesses places a heavy burden on clinical services in many low- and middle-income countries (LMICs). Bacterial and viral aetiologies of acute fevers are often clinically indistinguishable and, in the absence of diagnostic tests, the 'just-in-case' use of antibiotics by many health workers has become common practice, which has an impact on drug-resistant infections. Our study aims to answer the following question: in patients with undifferentiated febrile illness presenting to outpatient clinics/peripheral health centres in LMICs, can we demonstrate an improvement in clinical outcomes and reduce unnecessary antibiotic prescription over current practice by using a combination of simple, accurate diagnostic tests, clinical algorithms, and training and communication (intervention package)?MethodsWe designed a randomized, controlled clinical trial to evaluate the impact of our intervention package on clinical outcomes and antibiotic prescription rates in acute febrile illnesses. Available, point-of-care, pathogen-specific and non-pathogen specific (host markers), rapid diagnostic tests (RDTs) included in the intervention package were selected based on pre-defined criteria. Nine clinical study sites in six countries (Burkina Faso, Ghana, India, Myanmar, Nepal and Uganda), which represent heterogeneous outpatient care settings, were selected. We considered the expected seasonal variations in the incidence of acute febrile illnesses across all the sites by ensuring a recruitment period of 12 months. A master protocol was developed and adapted for country-specific ethical submissions. Diagnostic algorithms and choice of RDTs acknowledged current data on aetiologies of acute febrile illnesses in each country. We included a qualitative evaluation of drivers and/or deterrents of uptake of new diagnostics and antibiotic use for acute febrile illnesses. Sample size estimations were based on historical site data of antibiotic prescription practices for malarial and non-malarial acute fevers. Overall, 9 semi-independent studies will enrol a minimum of 21,876 patients and an aggregate data meta-analysis will be conducted on completion.DiscussionThis study is expected to generate vital evidence needed to inform policy decisions on the role of rapid diagnostic tests in the clinical management of acute febrile illnesses, with a view to controlling the rise of antimicrobial resistance in LMICs.Trial registrationClinicaltrials.gov NCT04081051 . Registered on 6 September 2019. Protocol version 1.4 dated 20 December 2019.
Chagas disease, caused by the protozoan Trypanosoma cruzi is a neglected tropical disease with high prevalence (5.7 million in Latin America, WHO 2015), significant burden, and significant morbimortality mostly due to severe heart disorders during the chronic phase of infection. Chagas disease is endemic in Latin America, and medical care for the disease is the major expense for Brazil's Universal Healthcare System (Sistema Único de Saúde (SUS). The efficacy of the available drugs benznidazole and nifurtimox are low for the chronic phase of Chagas disease, the phase in which most patients are diagnosed, and there are frequent side effects, and drug resistance occurs. The rapid deployment of new drug regimens that are effective for the chronic phase treatment is low-cost and less toxic than the currently available therapy, which is a global priority. Repurposing drugs already in clinical use with other combinations would be the fastest and safest strategy for treating Chagas disease patients. We hypothesize that the combined treatment using repurposing drugs with benznidazole will be more efficacious than benznidazole alone. This needs to be tested further both in vitro and in animal models to understand the efficacy of the treatment before performing human clinical trials. We further hypothesize that producing nanoparticle formulation of the drugs can reduce their toxicity and improve therapeutic use.
An observational cohort study on the incidence of SARS-CoV-2 infection and B.1.1.7 variant infection in healthcare workers by antibody and vaccination status.
Natural and vaccine-induced immunity will play a key role in controlling the SARS-CoV-2 pandemic. SARS-CoV-2 variants have the potential to evade natural and vaccine-induced immunity. In a longitudinal cohort study of healthcare workers (HCWs) in Oxfordshire, UK, we investigated the protection from symptomatic and asymptomatic PCR-confirmed SARS-CoV-2 infection conferred by vaccination (Pfizer-BioNTech BNT162b2, Oxford-AstraZeneca ChAdOx1 nCOV-19) and prior infection (determined using anti-spike antibody status), using Poisson regression adjusted for age, sex, temporal changes in incidence and role. We estimated protection conferred after one versus two vaccinations and from infections with the B.1.1.7 variant identified using whole genome sequencing. 13,109 HCWs participated; 8285 received the Pfizer-BioNTech vaccine (1407 two doses) and 2738 the Oxford-AstraZeneca vaccine (49 two doses). Compared to unvaccinated seronegative HCWs, natural immunity and two vaccination doses provided similar protection against symptomatic infection: no HCW vaccinated twice had symptomatic infection, and incidence was 98% lower in seropositive HCWs (adjusted incidence rate ratio 0.02 [95%CI <0.01-0.18]). Two vaccine doses or seropositivity reduced the incidence of any PCR-positive result with or without symptoms by 90% (0.10 [0.02-0.38]) and 85% (0.15 [0.08-0.26]) respectively. Single-dose vaccination reduced the incidence of symptomatic infection by 67% (0.33 [0.21-0.52]) and any PCR-positive result by 64% (0.36 [0.26-0.50]). There was no evidence of differences in immunity induced by natural infection and vaccination for infections with S-gene target failure and B.1.1.7. Natural infection resulting in detectable anti-spike antibodies and two vaccine doses both provide robust protection against SARS-CoV-2 infection, including against the B.1.1.7 variant.
AbstractPre-existing T cell immunity to SARS-CoV-2 in individuals without prior exposure to SARS-CoV-2 has been reported in several studies. While emerging evidence hints toward prior exposure to common-cold human coronaviruses (HCoV), the extent of- and conditions for-cross-protective immunity between SARS-CoV-2 and HCoVs remain open. Here, by leveraging a comprehensive pool of publicly available functionally evaluated SARS-CoV-2 peptides, we report 126 immunogenic SARS-CoV-2 peptides with high sequence similarity to 285 MHC-presented target peptides from at least one of four HCoV, thus providing a map describing the landscape of SARS-CoV-2 shared and private immunogenic peptides with functionally validated T cell responses. Using this map, we show that while SARS-CoV-2 immunogenic peptides in general exhibit higher level of dissimilarity to both self-proteome and -microbiomes, there exist several SARS-CoV-2 immunogenic peptides with high similarity to various human protein coding genes, some of which have been reported to have elevated expression in severe COVID-19 patients. We then combine our map with a SARS-CoV-2-specific TCR repertoire data from COVID-19 patients and healthy controls and show that whereas the public repertoire for the majority of convalescent patients are dominated by TCRs cognate to private SARS-CoV-2 peptides, for a subset of patients, more than 50% of their public repertoires that show reactivity to SARS-CoV-2, consist of TCRs cognate to shared SARS-CoV-2-HCoV peptides. Further analyses suggest that the skewed distribution of TCRs cognate to shared and private peptides in COVID-19 patients is likely to be HLA-dependent. Finally, by utilising the global prevalence of HLA alleles, we provide 10 peptides with known cognate TCRs that are conserved across SARS-CoV-2 and multiple human coronaviruses and are predicted to be recognised by a high proportion of the global population. Overall, our work indicates the potential for HCoV-SARS-CoV-2 reactive CD8+ T cells, which is likely dependent on differences in HLA-coding genes among individuals. These findings may have important implications for COVID-19 heterogeneity and vaccine-induced immune responses as well as robustness of immunity to SARS-CoV-2 and its variants.
Bacterial extracellular polysaccharides (EPSs) play critical roles in virulence. Many bacteria assemble EPSs via a multi-protein "Wzx-Wzy" system, involving glycan polymerization at the outer face of the cytoplasmic/inner membrane. Gram-negative species couple polymerization with translocation across the periplasm and outer membrane and the master regulator of the system is the tyrosine autokinase, Wzc. This near atomic cryo-EM structure of dephosphorylated Wzc from E. coli shows an octameric assembly with a large central cavity formed by transmembrane helices. The tyrosine autokinase domain forms the cytoplasm region, while the periplasmic region contains small folded motifs and helical bundles. The helical bundles are essential for function, most likely through interaction with the outer membrane translocon, Wza. Autophosphorylation of the tyrosine-rich C-terminus of Wzc results in disassembly of the octamer into multiply phosphorylated monomers. We propose that the cycling between phosphorylated monomer and dephosphorylated octamer regulates glycan polymerization and translocation.
Cancer cells exhibit an altered metabolic phenotype, consuming higher levels of the amino acid glutamine. This metabolic reprogramming depends on increased mitochondrial glutaminase activity to convert glutamine to glutamate, an essential precursor for bioenergetic and biosynthetic processes in cells. Mammals encode the kidney-type (GLS) and liver-type (GLS2) glutaminase isozymes. GLS is overexpressed in cancer and associated with enhanced malignancy. On the other hand, GLS2 is either a tumor suppressor or an oncogene, depending on the tumor type. The GLS structure and activation mechanism are well known, while the structural determinants for GLS2 activation remain elusive. Here, we describe the structure of the human glutaminase domain of GLS2, followed by the functional characterization of the residues critical for its activity. Increasing concentrations of GLS2 lead to tetramer stabilization, a process enhanced by phosphate. In GLS2, the so-called "lid loop" is in a rigid open conformation, which may be related to its higher affinity for phosphate and lower affinity for glutamine; hence, it has lower glutaminase activity than GLS. The lower affinity of GLS2 for glutamine is also related to its less electropositive catalytic site than GLS, as indicated by a Thr225Lys substitution within the catalytic site decreasing the GLS2 glutamine concentration corresponding to half-maximal velocity (K0.5). Finally, we show that the Lys253Ala substitution (corresponding to the Lys320Ala in the GLS "activation" loop, formerly known as the "gating" loop) renders a highly active protein in stable tetrameric form. We conclude that the "activation" loop, a known target for GLS inhibition, may also be a drug target for GLS2.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has undergone progressive change, with variants conferring advantage rapidly becoming dominant lineages, e.g., B.1.617. With apparent increased transmissibility, variant B.1.617.2 has contributed to the current wave of infection ravaging the Indian subcontinent and has been designated a variant of concern in the United Kingdom. Here we study the ability of monoclonal antibodies and convalescent and vaccine sera to neutralize B.1.617.1 and B.1.617.2, complement this with structural analyses of Fab/receptor binding domain (RBD) complexes, and map the antigenic space of current variants. Neutralization of both viruses is reduced compared with ancestral Wuhan-related strains, but there is no evidence of widespread antibody escape as seen with B.1.351. However, B.1.351 and P.1 sera showed markedly more reduction in neutralization of B.1.617.2, suggesting that individuals infected previously by these variants may be more susceptible to reinfection by B.1.617.2. This observation provides important new insights for immunization policy with future variant vaccines in non-immune populations.
Estimating the effectiveness of first dose of COVID-19 vaccine against mortality in England: a quasi-experimental study
AbstractBackgroundEstimating real-world vaccine effectiveness is vital to assess the impact of the vaccination programme on the pandemic and inform the ongoing policy response. However, estimating vaccine effectiveness using observational data is inherently challenging because of the non-randomised design and the potential for unmeasured confounding.MethodsWe used a Regression Discontinuity Design (RDD) to estimate vaccine effectiveness against COVID-19 mortality in England, exploiting the discontinuity in vaccination rates resulting from the UK’s age-based vaccination priority groups. We used the fact that people aged 80 or over were prioritised for the vaccine roll-out in the UK to compare the risk of COVID-19 and non-COVID-19 death in people aged 75–79 and 80–84.FindingsThe prioritisation of vaccination of people aged 80 or above led to a large discrepancy in vaccination rates in people 80–84 compared to those 75–79 at the beginning of the vaccination campaign. We found a corresponding difference in COVID-19 mortality, but not in non-COVID-19 mortality, suggesting that our approach appropriately addresses the issue of unmeasured confounding factors. Our results suggest that the first vaccine dose reduced the risk of COVID-19 death by 70.5% (95% CI 18.2–117.7) in those aged 80.InterpretationsOur results support existing evidence that a first dose of a COVID-19 vaccine has a strong protective effect against COVID-19 mortality in older adults. The RDD estimate of vaccine effectiveness is comparable to previously published studies using different methods, suggesting that unmeasured confounding factors are unlikely to substantially bias these studies.FundingOffice for National Statistics.Research in ContextEvidence before this studyWe searched PubMed for studies reporting on the ‘real-world’ effectiveness of the COVID-19 vaccination on risk of death using terms such as “COVID-19”, “vaccine effectiveness”, “mortality” and “death”. The relevant published studies on this topic report vaccine effectiveness estimates against risk of death ranging from 64.2% to 98.7%, for varying times post-vaccination. All of these are observational studies and therefore potentially subject to bias from unmeasured confounding.We found no studies that used a quasi-experimental method such as regression discontinuity design, which is not subject to bias from unmeasured confounding, to calculate the effectiveness of the COVID-19 vaccination on risk of COVID-19 death, or on other outcomes such as hospitalisation or infection.Added value of this studyThe estimates of vaccine effectiveness based on observational data may be biased by unmeasured confounding. This study uses a regression discontinuity design to estimate vaccine effectiveness, exploiting the fact that the vaccination campaign in the UK was rolled out following age-based priority groups. This enables the calculation of an unbiased estimate of the effectiveness of the COVID-19 vaccine against risk of death.The vaccine effectiveness estimate of 70.5% (95% CI 18.2–117.7) is similar to previously published estimates, therefore suggesting that these estimates are not substantially affected by unmeasured confounding factors and confirming the effectiveness of the COVID-19 vaccine against risk of COVID-19 death.Implications of all the available evidenceObtaining an unbiased estimate of COVID-19 vaccine effectiveness is of vital importance in informing policy for lifting COVID-19 related measures. The regression discontinuity design provides confidence that the existing estimates from observational studies are unlikely to be substantially biased by unmeasured confounding.
Nanopore metagenomic sequencing of influenza virus directly from respiratory samples: diagnosis, drug resistance and nosocomial transmission, United Kingdom, 2018/19 influenza season
Background Influenza virus presents a considerable challenge to public health by causing seasonal epidemics and occasional pandemics. Nanopore metagenomic sequencing has the potential to be deployed for near-patient testing, providing rapid infection diagnosis, rationalising antimicrobial therapy, and supporting infection-control interventions. Aim To evaluate the applicability of this sequencing approach as a routine laboratory test for influenza in clinical settings. Methods We conducted Oxford Nanopore Technologies (Oxford, United Kingdom (UK)) metagenomic sequencing for 180 respiratory samples from a UK hospital during the 2018/19 influenza season, and compared results to routine molecular diagnostic standards (Xpert Xpress Flu/RSV assay; BioFire FilmArray Respiratory Panel 2 assay). We investigated drug resistance, genetic diversity, and nosocomial transmission using influenza sequence data. Results Compared to standard testing, Nanopore metagenomic sequencing was 83% (75/90) sensitive and 93% (84/90) specific for detecting influenza A viruses. Of 59 samples with haemagglutinin subtype determined, 40 were H1 and 19 H3. We identified an influenza A(H3N2) genome encoding the oseltamivir resistance S331R mutation in neuraminidase, potentially associated with an emerging distinct intra-subtype reassortant. Whole genome phylogeny refuted suspicions of a transmission cluster in a ward, but identified two other clusters that likely reflected nosocomial transmission, associated with a predominant community-circulating strain. We also detected other potentially pathogenic viruses and bacteria from the metagenome. Conclusion Nanopore metagenomic sequencing can detect the emergence of novel variants and drug resistance, providing timely insights into antimicrobial stewardship and vaccine design. Full genome generation can help investigate and manage nosocomial outbreaks.
Chronic hepatitis B infection remains a serious global health threat, contributing to a large number of deaths through liver cirrhosis and hepatocellular carcinoma. Current treatment does not eradicate disease, and therefore new treatments are urgently needed. In acute hepatitis B virus (HBV) a strong immune response is necessary to clear the virus, but in chronic infection the immune response is weakened and dysfunctional. Therapeutic vaccination describes the process of inoculating individuals with a non-infective form of viral antigen with the aim of inducing or boosting existing HBV-specific immune responses, resulting in sustained control of HBV infection. In this review we outline the rationale for therapeutic vaccination in chronic HBV infection, discuss previous and ongoing trials of novel HBV therapeutic vaccine candidates and outline strategies to improve vaccine efficacy going forward.
Potassium-coupled chloride transporters (KCCs) play crucial roles in regulating cell volume and intracellular chloride concentration. They are characteristically inhibited under isotonic conditions via phospho-regulatory sites located within the cytoplasmic termini. Decreased inhibitory phosphorylation in response to hypotonic cell swelling stimulates transport activity, and dysfunction of this regulatory process has been associated with various human diseases. Here, we present cryo-EM structures of human KCC3b and KCC1, revealing structural determinants for phospho-regulation in both N- and C-termini. We show that phospho-mimetic KCC3b is arrested in an inward-facing state in which intracellular ion access is blocked by extensive contacts with the N-terminus. In another mutant with increased isotonic transport activity, KCC1Δ19, this interdomain interaction is absent, likely due to a unique phospho-regulatory site in the KCC1 N-terminus. Furthermore, we map additional phosphorylation sites as well as a previously unknown ATP/ADP-binding pocket in the large C-terminal domain and show enhanced thermal stabilization of other CCCs by adenine nucleotides. These findings provide fundamentally new insights into the complex regulation of KCCs and may unlock innovative strategies for drug development.
High cure rates for HCV genotype 6 in advanced liver fibrosis with 12 weeks sofosbuvir and daclatasvir: The Vietnam SEARCH Study
Abstract Background Genotype 6 is the most genetically diverse lineage of hepatitis C virus (HCV), and predominates in Vietnam. It can be treated with sofosbuvir with daclatasvir (SOF/DCV), the lowest costing treatment combination globally. In regional guidelines, longer treatment durations of SOF/DCV (24 weeks) are recommended for cirrhotic individuals, compared with other pangenotypic regimens (12 weeks), based on sparse data. Early on-treatment virological response may offer means of reducing length and cost of therapy in patients with liver fibrosis. Methods In this prospective trial in Vietnam, genotype 6-infected adults with advanced liver fibrosis or compensated cirrhosis were treated with SOF/DCV. Day 14 viral load was used to guide duration of therapy: participants with viral load <500 IU/ml at day 14 were treated with 12 weeks of SOF/DCV and those ≥500 IU/ml received 24 weeks. Primary endpoint was sustained virological response. Findings Of 41 individuals with advanced fibrosis or compensated cirrhosis who commenced treatment, 51% had genotype 6a, 34% 6e. The remainder had 6h, 6k, 6l or 6o. 100% had viral load <500 IU/ml by day 14, meaning all received 12 weeks of SOF/DCV. 100% achieved SVR12 despite a high frequency of putative NS5A inhibitor resistance-associated substitutions (RAS) at baseline. Interpretation 12 weeks of SOF/DCV achieves excellent cure rates in this population. This data supports the removal of costly genotyping in countries where genotype 3 prevalence in <5%, in keeping with WHO guidelines. NS5A-resistance associated mutations in isolation, do not affect efficacy of SOF/DCV therapy. Wider evaluation of response-guided therapy is warranted.