Building pyramids against the evolutionary emergence of pathogens.

Mutations allowing pathogens to escape host immunity promote the spread of infectious diseases in heterogeneous host populations and can lead to major epidemics. Understanding the conditions that slow down this evolution is key for the development of durable control strategies against pathogens. Here, we use theory and experiments to compare the efficacy of three strategies for the deployment of resistance: (i) a mixing strategy where the host population contains two single-resistant genotypes, (ii) a pyramiding strategy where the host carries a double-resistant genotype, (iii) a combining strategy where the host population is a mix of a single-resistant genotype and a double-resistant genotype. First, we use evolutionary epidemiology theory to clarify the interplay between demographic stochasticity and evolutionary dynamics to show that the pyramiding strategy always yields lower probability of evolutionary emergence. Second, we test experimentally these predictions with the introduction of bacteriophages into bacterial populations where we manipulated the diversity and the depth of immunity using a Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated (CRISPR-Cas) system. These biological assays confirm that pyramiding multiple defences into the same host genotype and avoiding combination with single-defence genotypes is a robust way to reduce pathogen evolutionary emergence. The experimental validation of these theoretical recommendations has practical implications in various areas, including for the optimal deployment of resistance varieties in agriculture and for the design of durable vaccination strategies.

Exploring indirect protection associated with influenza immunization - A systematic review of the literature.

BACKGROUND: Influenza causes significant annual morbidity and mortality, particularly in older adults, for whom influenza vaccine effectiveness (VE) is also lower. Immunizing one group (e.g., children) against influenza may indirectly protect another group (e.g., older adults) against influenza and its complications. METHODS: We updated previous systematic reviews on indirect protection against influenza by searching MEDLINE and EMBASE for relevant human studies published until January 4, 2017. We abstracted and critically appraised English language publications that reported or provided information to calculate indirect VE against influenza, as a percentage, in non-institutional settings. We developed a term called 'estimated actual protection' to explore the relationship between indirect protection and the product of direct VE and relative vaccine coverage. We calculated estimated actual protection for a subset of studies that reported coverage and indirect VE for: laboratory-confirmed influenza; outpatient care for respiratory illness; influenza-associated emergency visits; or influenza-associated hospitalizations. We ran linear mixed models to compare estimated actual protection against indirect VE for the four outcomes, and graphed the data. RESULTS: Of 2320 unique records identified, we abstracted and appraised 26 articles describing 24 studies. The majority of included studies reported at least one outcome suggesting that immunizing one group reduced influenza-related outcomes in another group. Critical appraisal of the abstracted studies identified recurring methodological weaknesses, such as lack of laboratory-confirmed influenza. Our exploratory analyses of 18 studies indicated a positive but not statistically significant relationship between estimated actual protection and indirect protection for each of the four outcomes. CONCLUSIONS: Our systematic review and exploratory analyses suggest influenza immunization provides some level of indirect protection. However, our critical appraisal highlights the need for a standardized and consistently applied approach to measuring indirect protection against influenza to fill existing knowledge gaps. Additionally, the concept of estimated actual protection requires validation.

Equity and impact: Ontario's infant rotavirus immunization program five years following implementation. A population-based cohort study.

BACKGROUND: Ontario implemented a publicly-funded rotavirus (RV) immunization program in 2011. Our objectives were to evaluate its impact on hospitalizations and emergency department (ED) visits for acute gastroenteritis (AGE) five years after implementation. METHODS: We performed a population-based longitudinal retrospective cohort study to identify hospitalizations and ED visits for RV-AGE and overall AGE in all age groups using ICD-10 codes between August 1, 2005 and March 31, 2016. A negative binomial regression model that included the effect of time was used to calculate rates, rate ratios (RRs) and 95% confidence intervals (CIs) for AGE before and after the program's implementation, after adjusting for age, seasonality and secular trends. We examined the seasonality of RV-AGE hospitalizations among children under five before and after the program and explored its equity impact. RESULTS: Following program implementation, RV-AGE hospitalizations and ED visits among children under five years declined by 76% (RR 0.24, 95% CI 0.20-0.28) and 68% (RR 0.32, 95% CI 0.21-0.50), respectively. In addition, hospitalizations and ED visits for overall AGE declined by 38% (RR 0.62, 95% CI 0.59-0.65) and 26% (RR 0.74, 95% CI 0.73-0.76), respectively, among children under age five. Significant reductions in both outcomes were also found across a range of age-strata. In the pre-program period, the mean monthly hospitalization rate for RV-AGE among children residing in the most marginalized neighbourhoods was 33% higher than those residing in the least marginalized (RR 1.33, 95% CI 1.17-1.52), this disparity was not evident in the program period (RR 0.95, 95% CI 0.69-1.32). We found no evidence of a seasonal shift in rotavirus pediatric hospitalizations. INTERPRETATION: The introduction of routine infant rotavirus immunization has had a substantial population impact in Ontario. Our study confirms herd effects and suggests the program may have reduced previous inequities in the burden of pediatric rotavirus hospitalizations.

Targeted Genome Editing of Virulent Phages Using CRISPR-Cas9.

This protocol describes a straightforward method to generate specific mutations in the genome of strictly lytic phages. Briefly, a targeting CRISPR-Cas9 system and a repair template suited for homologous recombination are provided inside a bacterial host, here the Gram-positive model Lactococcus lactis MG1363. The CRISPR-Cas9 system is programmed to cleave a specific region present on the genome of the invading phage, but absent from the recombination template. The system either triggers the recombination event or exerts the selective pressure required to isolate recombinant phages. With this methodology, we generated multiple gene knockouts, a point mutation and an insertion in the genome of the virulent lactococcal phage p2. Considering the broad host range of the plasmids used in this protocol, the latter can be extrapolated to other phage-host pairs.