Methodological challenges in European ethics approvals for a genetic epidemiology study in critically ill patients: the GenOSept experience.

BACKGROUND: During the set-up phase of an international study of genetic influences on outcomes from sepsis, we aimed to characterise potential differences in ethics approval processes and outcomes in participating European countries. METHODS: Between 2005 and 2007 of the FP6-funded international Genetics Of Sepsis and Septic Shock (GenOSept) project, we asked national coordinators to complete a structured survey of research ethic committee (REC) approval structures and processes in their countries, and linked these data to outcomes. Survey findings were reconfirmed or modified in 2017. RESULTS: Eighteen countries participated in the study, recruiting 2257 patients from 160 ICUs. National practices differed widely in terms of composition of RECs, procedures and duration of the ethics approval process. Eight (44.4%) countries used a single centralised process for approval, seven (38.9%) required approval by an ethics committee in each participating hospital, and three (16.7%) required both. Outcomes of the application process differed widely between countries because of differences in national legislation, and differed within countries because of interpretation of the ethics of conducting research in patients lacking capacity. The RECs in four countries had no lay representation. The median time from submission to final decision was 1.5 (interquartile range 1-7) months; in nine (50%) approval was received within 1 month; six took over 6 months, and in one 24 months; had all countries been able to match the most efficient approvals processes, an additional 74 months of country or institution-level recruitment would have been available. In three countries, rejection of the application by some local RECs resulted in loss of centres; and one country rejected the application outright. CONCLUSIONS: The potential benefits of the single application portal offered by the European Clinical Trials Regulation will not be realised without harmonisation of research ethics committee practices as well as national legislation.

SCOPA and META-SCOPA: software for the analysis and aggregation of genome-wide association studies of multiple correlated phenotypes.

BACKGROUND: Genome-wide association studies (GWAS) of single nucleotide polymorphisms (SNPs) have been successful in identifying loci contributing genetic effects to a wide range of complex human diseases and quantitative traits. The traditional approach to GWAS analysis is to consider each phenotype separately, despite the fact that many diseases and quantitative traits are correlated with each other, and often measured in the same sample of individuals. Multivariate analyses of correlated phenotypes have been demonstrated, by simulation, to increase power to detect association with SNPs, and thus may enable improved detection of novel loci contributing to diseases and quantitative traits. RESULTS: We have developed the SCOPA software to enable GWAS analysis of multiple correlated phenotypes. The software implements "reverse regression" methodology, which treats the genotype of an individual at a SNP as the outcome and the phenotypes as predictors in a general linear model. SCOPA can be applied to quantitative traits and categorical phenotypes, and can accommodate imputed genotypes under a dosage model. The accompanying META-SCOPA software enables meta-analysis of association summary statistics from SCOPA across GWAS. Application of SCOPA to two GWAS of high-and low-density lipoprotein cholesterol, triglycerides and body mass index, and subsequent meta-analysis with META-SCOPA, highlighted stronger association signals than univariate phenotype analysis at established lipid and obesity loci. The META-SCOPA meta-analysis also revealed a novel signal of association at genome-wide significance for triglycerides mapping to GPC5 (lead SNP rs71427535, p = 1.1x10-8), which has not been reported in previous large-scale GWAS of lipid traits. CONCLUSIONS: The SCOPA and META-SCOPA software enable discovery and dissection of multiple phenotype association signals through implementation of a powerful reverse regression approach.

A flexible approach for the analysis of rare variants allowing for a mixture of effects on binary or quantitative traits.

Multiple rare variants either within or across genes have been hypothesised to collectively influence complex human traits. The increasing availability of high throughput sequencing technologies offers the opportunity to study the effect of rare variants on these traits. However, appropriate and computationally efficient analytical methods are required to account for collections of rare variants that display a combination of protective, deleterious and null effects on the trait. We have developed a novel method for the analysis of rare genetic variation in a gene, region or pathway that, by simply aggregating summary statistics at each variant, can: (i) test for the presence of a mixture of effects on a trait; (ii) be applied to both binary and quantitative traits in population-based and family-based data; (iii) adjust for covariates to allow for non-genetic risk factors and; (iv) incorporate imputed genetic variation. In addition, for preliminary identification of promising genes, the method can be applied to association summary statistics, available from meta-analysis of published data, for example, without the need for individual level genotype data. Through simulation, we show that our method is immune to the presence of bi-directional effects, with no apparent loss in power across a range of different mixtures, and can achieve greater power than existing approaches as long as summary statistics at each variant are robust. We apply our method to investigate association of type-1 diabetes with imputed rare variants within genes in the major histocompatibility complex using genotype data from the Wellcome Trust Case Control Consortium.

Genetic determinants of anti-malarial acquired immunity in a large multi-centre study.

BACKGROUND: Many studies report associations between human genetic factors and immunity to malaria but few have been reliably replicated. These studies are usually country-specific, use small sample sizes and are not directly comparable due to differences in methodologies. This study brings together samples and data collected from multiple sites across Africa and Asia to use standardized methods to look for consistent genetic effects on anti-malarial antibody levels. METHODS: Sera, DNA samples and clinical data were collected from 13,299 individuals from ten sites in Senegal, Mali, Burkina Faso, Sudan, Kenya, Tanzania, and Sri Lanka using standardized methods. DNA was extracted and typed for 202 Single Nucleotide Polymorphisms with known associations to malaria or antibody production, and antibody levels to four clinical grade malarial antigens [AMA1, MSP1, MSP2, and (NANP)4] plus total IgE were measured by ELISA techniques. Regression models were used to investigate the associations of clinical and genetic factors with antibody levels. RESULTS: Malaria infection increased levels of antibodies to malaria antigens and, as expected, stable predictors of anti-malarial antibody levels included age, seasonality, location, and ethnicity. Correlations between antibodies to blood-stage antigens AMA1, MSP1 and MSP2 were higher between themselves than with antibodies to the (NANP)4 epitope of the pre-erythrocytic circumsporozoite protein, while there was little or no correlation with total IgE levels. Individuals with sickle cell trait had significantly lower antibody levels to all blood-stage antigens, and recessive homozygotes for CD36 (rs321198) had significantly lower anti-malarial antibody levels to MSP2. CONCLUSION: Although the most significant finding with a consistent effect across sites was for sickle cell trait, its effect is likely to be via reducing a microscopically positive parasitaemia rather than directly on antibody levels. However, this study does demonstrate a framework for the feasibility of combining data from sites with heterogeneous malaria transmission levels across Africa and Asia with which to explore genetic effects on anti-malarial immunity.

Association between trends in clinical variables and outcome in intensive care patients with faecal peritonitis: analysis of the GenOSept cohort.

INTRODUCTION: Patients admitted to intensive care following surgery for faecal peritonitis present particular challenges in terms of clinical management and risk assessment. Collaborating surgical and intensive care teams need shared perspectives on prognosis. We aimed to determine the relationship between dynamic assessment of trends in selected variables and outcomes. METHODS: We analysed trends in physiological and laboratory variables during the first week of intensive care unit (ICU) stay in 977 patients at 102 centres across 16 European countries. The primary outcome was 6-month mortality. Secondary endpoints were ICU, hospital and 28-day mortality. For each trend, Cox proportional hazards (PH) regression analyses, adjusted for age and sex, were performed for each endpoint. RESULTS: Trends over the first 7 days of the ICU stay independently associated with 6-month mortality were worsening thrombocytopaenia (mortality: hazard ratio (HR) = 1.02; 95% confidence interval (CI), 1.01 to 1.03; P < 0.001) and renal function (total daily urine output: HR =1.02; 95% CI, 1.01 to 1.03; P < 0.001; Sequential Organ Failure Assessment (SOFA) renal subscore: HR = 0.87; 95% CI, 0.75 to 0.99; P = 0.047), maximum bilirubin level (HR = 0.99; 95% CI, 0.99 to 0.99; P = 0.02) and Glasgow Coma Scale (GCS) SOFA subscore (HR = 0.81; 95% CI, 0.68 to 0.98; P = 0.028). Changes in renal function (total daily urine output and renal component of the SOFA score), GCS component of the SOFA score, total SOFA score and worsening thrombocytopaenia were also independently associated with secondary outcomes (ICU, hospital and 28-day mortality). We detected the same pattern when we analysed trends on days 2, 3 and 5. Dynamic trends in all other measured laboratory and physiological variables, and in radiological findings, changes in respiratory support, renal replacement therapy and inotrope and/or vasopressor requirements failed to be retained as independently associated with outcome in multivariate analysis. CONCLUSIONS: Only deterioration in renal function, thrombocytopaenia and SOFA score over the first 2, 3, 5 and 7 days of the ICU stay were consistently associated with mortality at all endpoints. These findings may help to inform clinical decision making in patients with this common cause of critical illness.

Gene-lifestyle interaction and type 2 diabetes: the EPIC interact case-cohort study.

BACKGROUND: Understanding of the genetic basis of type 2 diabetes (T2D) has progressed rapidly, but the interactions between common genetic variants and lifestyle risk factors have not been systematically investigated in studies with adequate statistical power. Therefore, we aimed to quantify the combined effects of genetic and lifestyle factors on risk of T2D in order to inform strategies for prevention. METHODS AND FINDINGS: The InterAct study includes 12,403 incident T2D cases and a representative sub-cohort of 16,154 individuals from a cohort of 340,234 European participants with 3.99 million person-years of follow-up. We studied the combined effects of an additive genetic T2D risk score and modifiable and non-modifiable risk factors using Prentice-weighted Cox regression and random effects meta-analysis methods. The effect of the genetic score was significantly greater in younger individuals (p for interaction  = 1.20×10-4). Relative genetic risk (per standard deviation [4.4 risk alleles]) was also larger in participants who were leaner, both in terms of body mass index (p for interaction  = 1.50×10-3) and waist circumference (p for interaction  = 7.49×10-9). Examination of absolute risks by strata showed the importance of obesity for T2D risk. The 10-y cumulative incidence of T2D rose from 0.25% to 0.89% across extreme quartiles of the genetic score in normal weight individuals, compared to 4.22% to 7.99% in obese individuals. We detected no significant interactions between the genetic score and sex, diabetes family history, physical activity, or dietary habits assessed by a Mediterranean diet score. CONCLUSIONS: The relative effect of a T2D genetic risk score is greater in younger and leaner participants. However, this sub-group is at low absolute risk and would not be a logical target for preventive interventions. The high absolute risk associated with obesity at any level of genetic risk highlights the importance of universal rather than targeted approaches to lifestyle intervention.

Patients with community acquired pneumonia admitted to European intensive care units: an epidemiological survey of the GenOSept cohort.

INTRODUCTION: Community acquired pneumonia (CAP) is the most common infectious reason for admission to the Intensive Care Unit (ICU). The GenOSept study was designed to determine genetic influences on sepsis outcome. Phenotypic data was recorded using a robust clinical database allowing a contemporary analysis of the clinical characteristics, microbiology, outcomes and independent risk factors in patients with severe CAP admitted to ICUs across Europe. METHODS: Kaplan-Meier analysis was used to determine mortality rates. A Cox Proportional Hazards (PH) model was used to identify variables independently associated with 28-day and six-month mortality. RESULTS: Data from 1166 patients admitted to 102 centres across 17 countries was extracted. Median age was 64 years, 62% were male. Mortality rate at 28 days was 17%, rising to 27% at six months. Streptococcus pneumoniae was the commonest organism isolated (28% of cases) with no organism identified in 36%. Independent risk factors associated with an increased risk of death at six months included APACHE II score (hazard ratio, HR, 1.03; confidence interval, CI, 1.01-1.05), bilateral pulmonary infiltrates (HR1.44; CI 1.11-1.87) and ventilator support (HR 3.04; CI 1.64-5.62). Haematocrit, pH and urine volume on day one were all associated with a worse outcome. CONCLUSIONS: The mortality rate in patients with severe CAP admitted to European ICUs was 27% at six months. Streptococcus pneumoniae was the commonest organism isolated. In many cases the infecting organism was not identified. Ventilator support, the presence of diffuse pulmonary infiltrates, lower haematocrit, urine volume and pH on admission were independent predictors of a worse outcome.

A comparison of synthetic control approaches for the evaluation of policy interventions using observational data: Evaluating the impact of redesigning urgent and emergency care in Northumberland.

OBJECTIVE: To compare the original synthetic control (OSC) method with alternative approaches (Generalized [GSC], Micro [MSC], and Bayesian [BSC] synthetic control methods) and re-evaluate the impact of a significant restructuring of urgent and emergency care in Northeast England, which included the opening of the UK's first purpose-built specialist emergency care hospital. DATA SOURCES: Simulations and data from Secondary Uses Service data, a single comprehensive repository for patient-level health care data in England. STUDY DESIGN: Hospital use of individuals exposed and unexposed to the restructuring is compared. We estimate the impact using OSC, MSC, BSC, and GSC applied at the general practice level. We contrast the estimation methods' performance in a Monte Carlo simulation study. DATA COLLECTION/EXTRACTION METHODS: Hospital activity data from Secondary Uses Service for patients aged over 18 years registered at a general practice in England from April 2011 to March 2019. PRINCIPAL FINDINGS: None of the methods dominated all simulation scenarios. GSC was generally preferred. In contrast to an earlier evaluation that used OSC, GSC reported a smaller impact of the opening of the hospital on Accident and Emergency (A&E) department (also known as emergency department or casualty) visits and no evidence for any impact on the proportion of A&E patients seen within 4 h. CONCLUSIONS: The simulation study highlights cases where the considered methods may lead to biased estimates in health policy evaluations. GSC was found to be the most reliable method of those considered. Considering more disaggregated data over a longer time span and applying GSC indicates that the specialist emergency care hospitals in Northumbria had less impact on A&E visits and waiting times than suggested by the original evaluation which applied OSC to more aggregated data.

How is primary care access changing? A retrospective, repeated cross-sectional study of patient-initiated demand at general practices in England using a modern access model, 2019-2022.

OBJECTIVES: To explore trends in patient-initiated requests for general practice services and the association between patient characteristics including demographics, preferences for care and clinical needs and modes of patient contact (online vs telephone), and care delivery (face-to-face vs remote) at practices using a modern access model. DESIGN: Retrospective repeated cross-sectional study spanning March 2019 to February 2022. SETTING: General practices in England using the askmyGP online consultation system to implement a modern general practice access model using digital and non-digital (multimodal) access pathways and digitally supported triage to manage patient-initiated requests. PARTICIPANTS: 10 435 465 patient-initiated requests from 1 488 865 patients at 154 practices. RESULTS: Most requests were initiated online (72.1% in 2021/2022) rather than by telephone. Online users were likely to be female, younger than 45 years, asking about existing medical problems, had used the system before and frequent attenders (familiar patients). During the pandemic, request rates for face-to-face consultations fell while those for telephone consultations and online messages increased, with telephone consultations being most popular (53.8% in 2021/2022). Video was seldom requested. More than 60% of requests were consistently delivered in the mode requested. Face-to-face consultations were more likely to be used for the youngest and oldest patients, new medical problems, non-frequent attenders (unfamiliar patients) and those who requested a face-to-face consultation. Over the course of the study, request rates for patients aged over 44 years increased, for example, by 15.4% (p<0.01) for patients aged over 74 years. Rates for younger patients decreased by 32.6% (p<0.001) in 2020/2021, compared with 2019/2020, before recovering to prepandemic levels in 2021/2022. CONCLUSIONS: Demand patterns shed light on the characteristics of patients making requests for general practice services and the composition of the care backlog with implications for policy and practice. A modern general practice access model can be used effectively to manage patient-initiated demand.

Antibiotic prescribing in remote versus face-to-face consultations for acute respiratory infections in primary care in England: an observational study using target maximum likelihood estimation.

Background: The COVID-19 pandemic has led to an ongoing increase in the use of remote consultations in general practice in England. Although the evidence is limited, there are concerns that the increase in remote consultations could lead to more antibiotic prescribing. Methods: In this cohort study, we used patient-level primary care data from the Clinical Practice Research Datalink to estimate the association between consultation mode (remote versus face-to-face) and antibiotic prescribing in England for acute respiratory infections (ARI) between April 2021 and March 2022. Eligibility criteria were applied at both practice-level and patient-level. 400 practices in England were sampled at random and then 600,000 patients were randomly sampled from the eligible patients (whose sex was recorded). Consultations for acute respiratory infections were identified. All antibiotic prescriptions were included, with the exception of antituberculosis drugs and antileprotic drugs, as identified through chapter 5.1 of the British National Formulary. The CPRD Aurum data was linked to the COVID-19 ONS infection survey by region. All analyses were done at the individual level. Repeated consultations from the same patient within 7 days were grouped together. We used targeted maximum likelihood estimation, a causal machine learning method with adjustment for infection type and patient-level, clinician-level and practice-level factors. Findings: There were 45,997 ARI consultations (34,555 unique patients) within the study period, of which 28,127 were remote and 17,870 were face-to-face. For children, 48% of consultations were remote and, for adults, 66% were remote. For children, 42% of remote and 43% of face-to-face consultations led to an antibiotic prescription; the equivalent values for adults were 52% and 42%, respectively. After adjustment with TMLE, adults with a remote consultation had 23% (odds ratio [OR] 1.23, 95% CI: 1.18-1.29) higher chance of being prescribed antibiotics than if they had been seen face-to-face. We found no significant association between consultation mode and antibiotic prescribing in children (OR 1.04 95% CI: 0.98-1.11). Interpretation: The higher rates of antibiotic prescribing in remote consultations for adults are cause for concern. We see no significant difference in antibiotic prescribing between consultation mode for children. These findings should inform antimicrobial stewardship activities for health-care professionals and policy makers. Future research should examine differences in guideline-compliance between remote and face-to-face consultations to understand the factors driving antibiotic prescribing in different consultation modes. Funding: None.

Continuity of care and consultation mode in general practice: a cross-sectional and longitudinal study using patient-level and practice-level data from before and during the COVID-19 pandemic in England.

OBJECTIVES: Investigate trends in continuity of care with a general practitioner (GP) before and during the COVID-19 pandemic. Identify whether continuity of care is associated with consultation mode, controlling for other patient and practice characteristics. DESIGN: Retrospective cross-sectional and longitudinal observational studies. SETTING: Primary care records from 389 general practices participating in Clinical Practice Research Datalink Aurum in England. PARTICIPANTS: In the descriptive analysis, 100 000+ patients were included each month between April 2018 and April 2021. Modelling of the association between continuity of care and consultation mode focused on 153 475 and 125 298 patients in index months of February 2020 (before the pandemic) and February 2021 (during the pandemic) respectively, and 76 281 patients in both index months. PRIMARY AND SECONDARY OUTCOMES MEASURES: The primary outcome measure was the Usual Provider of Care index. Secondary outcomes included the Bice-Boxerman index and count of consultations with the most frequently seen GP. RESULTS: Continuity of care was gradually declining before the pandemic but stabilised during it. There were consistent demographic, socioeconomic and regional differences in continuity of care. An average of 23% of consultations were delivered remotely in the year to February 2020 compared with 76% in February 2021. We found little evidence consultation mode was associated with continuity at the patient level, controlling for a range of covariates. In contrast, patient characteristics and practice-level supply and demand were associated with continuity. CONCLUSIONS: We set out to examine the association of consultation mode with continuity of care but found that GP supply and patient demand were much more important. To improve continuity for patients, primary care capacity needs to increase. This requires sufficient, long-term investment in clinicians, staff, facilities and digital infrastructure. General practice also needs to transform ways of working to ensure continuity for those that need it, even in a capacity-constrained environment.

Consultation Rate and Mode by Deprivation in English General Practice From 2018 to 2022: Population-Based Study.

BACKGROUND: The COVID-19 pandemic has had a significant impact on primary care service delivery with an increased use of remote consultations. With general practice delivering record numbers of appointments and rising concerns around access, funding, and staffing in the UK National Health Service, we assessed contemporary trends in consultation rate and modes (ie, face-to-face versus remote). OBJECTIVE: This paper describes trends in consultation rates in general practice in England for key demographics before and during the COVID-19 pandemic. We explore the use of remote and face-to-face consultations with regard to socioeconomic deprivation to understand the possible effect of changes in consultation modes on health inequalities. METHODS: We did a retrospective analysis of 9,429,919 consultations by general practitioners, nurses, or other health care professionals between March 2018 and February 2022 for patients registered at 397 general practices in England. We used routine electronic health records from Clinical Practice Research Datalink Aurum with linkage to national data sets. Negative binomial models were used to predict consultation rates and modes (ie, remote versus face-to-face) by age, sex, and socioeconomic deprivation over time. RESULTS: Overall consultation rates increased by 15% from 4.92 in 2018-2019 to 5.66 in 2021-2022 with some fluctuation during the start of the COVID-19 pandemic. The breakdown into face-to-face and remote consultations shows that the pandemic precipitated a rapid increase in remote consultations across all groups, but the extent varies by age. Consultation rates increased with increasing levels of deprivation. Socioeconomic differences in consultation rates, adjusted for sex and age, halved during the pandemic (from 0.36 to 0.18, indicating more consultations in the most deprived), effectively narrowing relative differences between deprivation quintiles. This trend remains when stratified by sex, but the difference across deprivation quintiles is smaller for men. The most deprived saw a relatively larger increase in remote and decrease in face-to-face consultation rates compared to the least deprived. CONCLUSIONS: The substantial increases in consultation rates observed in this study imply an increased pressure on general practice. The narrowing of consultation rates between deprivation quintiles is cause for concern, given ample evidence that health needs are greater in more deprived areas.

Aspects of observing and claiming allele flips in association studies.

Significant allele flipping, where associations for the same disease occur at opposite alleles of the same bi-allelic locus, is increasing. But when is a significant allele flip genuine? We address the statistical issues of claiming and observing genuine allele flips in actual samples. We show that unless an allele flip is genuine, the probability of observing a significant allele flip in samples ascertained similarly from a common population is negligible. We derive expressions for the expected values of commonly used measures of association, which confirm previous findings that the underlying mechanism of a genuine allele flip is variation in the haplotype frequencies and show further how this variation interacts with variation in the genetic effects to impact allele flipping. We show that for association testing at proxy SNPs, common in genome-wide association studies, variation in haplotype frequencies must coincide with a reversal in the sign of linkage disequilibrium (LD) to trigger genuine allele flips. Using HapMap data and r, rather than r(2), to highlight previously unobserved effects, we show that unless genetic effects are large, variation in LD is unlikely to cause genuine allele flips in samples drawn from the same population. However, as populations diverge, it is an increasingly viable cause of a genuine allele flip for sufficiently large genetic effect and/or sample sizes. We conclude that evidence of variation in local patterns of LD, ancestral composition of study samples, and environmental exposures between study populations can provide compelling practical evidence in defense of a genuine allele flip.

The challenge of detecting epistasis (G x G interactions): Genetic Analysis Workshop 16.

Interest is increasing in epistasis as a possible source of the unexplained variance missed by genome-wide association studies. The Genetic Analysis Workshop 16 Group 9 participants evaluated a wide variety of classical and novel analytical methods for detecting epistasis, in both the statistical and machine learning paradigms, applied to both real and simulated data. Because the magnitude of epistasis is clearly relative to scale of penetrance, and therefore to some extent, to the choice of model framework, it is not surprising that strong interactions under one model might be minimized or even disappear entirely under a different modeling framework.

A comparison of sample size and power in case-only association studies of gene-environment interaction.

Assuming continuous, normally distributed environmental and categorical genotype variables, the authors compare 6 case-only designs for tests of association in gene-environment interaction. Novel tests modeling the environmental variable as either the response or the predictor and allowing a genetic variable with multiallelic variants are included. The authors show that tests imposing the same genotypic pattern of inheritance perform similarly regardless of whether genotype is the response variable or the predictor variable. The novel tests using the genetic variable as the response variable are advantageous because they are robust to non-normally distributed environmental exposures. Dominance deviance-deviation from additivity in the main or interaction effects-is key to test performance: When it is zero or modest, tests searching for a trend with increasing risk alleles are optimal; when it is large, tests for genotypic effects are optimal. However, the authors show that dominance deviance is attenuated when it is observed at a proxy locus, which is common in genome-wide association studies, so large dominance deviance is likely to be rare. The authors conclude that the trend test is the appropriate tool for large-scale association scans where the true gene-environment interaction model is unknown. The common practice of assuming a dominant pattern of inheritance can cause serious losses of power in the presence of any recessive, or modest dominant, effects.

Derivation and validation of a prognostic model for postoperative risk stratification of critically ill patients with faecal peritonitis.

BACKGROUND: Prognostic scores and models of illness severity are useful both clinically and for research. The aim of this study was to develop two prognostic models for the prediction of long-term (6 months) and 28-day mortality of postoperative critically ill patients with faecal peritonitis (FP). METHODS: Patients admitted to intensive care units with faecal peritonitis and recruited to the European GenOSept study were divided into a derivation and a geographical validation subset; patients subsequently recruited to the UK GAinS study were used for temporal validation. Using all 50 clinical and laboratory variables available on day 1 of critical care admission, Cox proportional hazards regression was fitted to select variables for inclusion in two prognostic models, using stepwise selection and nonparametric bootstrapping sampling techniques. Using Area under the receiver operating characteristic curve (AuROC) analysis, the performance of the models was compared to SOFA and APACHE II. RESULTS: Five variables (age, SOFA score, lowest temperature, highest heart rate, haematocrit) were entered into the prognostic models. The discriminatory performance of the 6-month prognostic model yielded an AuROC 0.81 (95% CI 0.76-0.86), 0.73 (95% CI 0.69-0.78) and 0.76 (95% CI 0.69-0.83) for the derivation, geographic and temporal external validation cohorts, respectively. The 28-day prognostic tool yielded an AuROC 0.82 (95% CI 0.77-0.88), 0.75 (95% CI 0.69-0.80) and 0.79 (95% CI 0.71-0.87) for the same cohorts. These AuROCs appeared consistently superior to those obtained with the SOFA and APACHE II scores alone. CONCLUSIONS: The two prognostic models developed for 6-month and 28-day mortality prediction in critically ill septic patients with FP, in the postoperative phase, enhanced the day one SOFA score's predictive utility by adding a few key variables: age, lowest recorded temperature, highest recorded heart rate and haematocrit. External validation of their predictive capability in larger cohorts is needed, before introduction of the proposed scores into clinical practice to inform decision making and the design of clinical trials.

Resistance to malaria through structural variation of red blood cell invasion receptors.

The malaria parasite Plasmodium falciparum invades human red blood cells by a series of interactions between host and parasite surface proteins. By analyzing genome sequence data from human populations, including 1269 individuals from sub-Saharan Africa, we identify a diverse array of large copy-number variants affecting the host invasion receptor genes GYPA and GYPB We find that a nearby association with severe malaria is explained by a complex structural rearrangement involving the loss of GYPB and gain of two GYPB-A hybrid genes, which encode a serologically distinct blood group antigen known as Dantu. This variant reduces the risk of severe malaria by 40% and has recently increased in frequency in parts of Kenya, yet it appears to be absent from west Africa. These findings link structural variation of red blood cell invasion receptors with natural resistance to severe malaria.

Characterisation of the opposing effects of G6PD deficiency on cerebral malaria and severe malarial anaemia.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is believed to confer protection against Plasmodium falciparum malaria, but the precise nature of the protective effecthas proved difficult to define as G6PD deficiency has multiple allelic variants with different effects in males and females, and it has heterogeneous effects on the clinical outcome of P. falciparum infection. Here we report an analysis of multiple allelic forms of G6PD deficiency in a large multi-centre case-control study of severe malaria, using the WHO classification of G6PD mutations to estimate each individual's level of enzyme activity from their genotype. Aggregated across all genotypes, we find that increasing levels of G6PD deficiency are associated with decreasing risk of cerebral malaria, but with increased risk of severe malarial anaemia. Models of balancing selection based on these findings indicate that an evolutionary trade-off between different clinical outcomes of P. falciparum infection could have been a major cause of the high levels of G6PD polymorphism seen in human populations.

Disentangling linkage disequilibrium and linkage from dense single-nucleotide polymorphism trio data.

Parent-offspring trios are widely collected for disease gene-mapping studies and are being extensively genotyped as part of the International HapMap Project. With dense maps of markers on trios, the effects of LD and linkage can be separated, allowing estimation of recombination rates in a model-free setting. Here we define a model-free multipoint method on the basis of dense sequence polymorphism data from parent-offspring trios to estimate intermarker recombination rates. We use simulations to show that this method has up to 92% power to detect recombination hotspots of intensity 25 times background over a region of size 10 kb typed at density 1 marker per 2.5 kb and almost 100% power to detect large hotspots of intensity >125 times background over regions of size 10 kb typed with just 1 marker per 5 kb (alpha = 0.05). We found strong agreement at megabase scales between estimates from our method applied to HapMap trio data and estimates from the genetic map. At finer scales, using Centre d'Etude du Polymorphisme Humain (CEPH) pedigree data across a 10-Mb region of chromosome 20, a comparison of population recombination rate estimates obtained from our method with estimates obtained using a coalescent-based approximate-likelihood method implemented in PHASE 2.0 shows detection of the same coldspots and most hotspots: The Spearman rank correlation between the estimates from our method and those from PHASE is 0.58 (p < 2.2(-16)).

Glucose-6-phosphate dehydrogenase deficiency and the risk of malaria and other diseases in children in Kenya: a case-control and a cohort study.

BACKGROUND: The global prevalence of X-linked glucose-6-phosphate dehydrogenase (G6PD) deficiency is thought to be a result of selection by malaria, but epidemiological studies have yielded confusing results. We investigated the relationships between G6PD deficiency and both malaria and non-malarial illnesses among children in Kenya. METHODS: We did this study in Kilifi County, Kenya, where the G6PD c.202T allele is the only significant cause of G6PD deficiency. We tested the associations between G6PD deficiency and severe and complicated Plasmodium falciparum malaria through a case-control study of 2220 case and 3940 control children. Cases were children aged younger than 14 years, who visited the high dependency ward of Kilifi County Hospital with severe malaria between March 1, 1998, and Feb 28, 2010. Controls were children aged between 3-12 months who were born within the same study area between August 2006, and September 2010. We assessed the association between G6PD deficiency and both uncomplicated malaria and other common diseases of childhood in a cohort study of 752 children aged younger than 10 years. Participants of this study were recruited from a representative sample of households within the Ngerenya and Chonyi areas of Kilifi County between Aug 1, 1998, and July 31, 2001. The primary outcome measure for the case-control study was the odds ratio for hospital admission with severe malaria (computed by logistic regression) while for the cohort study it was the incidence rate ratio for uncomplicated malaria and non-malaria illnesses (computed by Poisson regression), by G6PD deficiency category. FINDINGS: 2863 (73%) children in the control group versus 1643 (74%) in the case group had the G6PD normal genotype, 639 (16%) versus 306 (14%) were girls heterozygous for G6PD c.202T, and 438 (11%) versus 271 (12%) children were either homozygous girls or hemizygous boys. Compared with boys and girls without G6PD deficiency, we found significant protection from severe malaria (odds ratio [OR] 0·82, 95% CI 0·70-0·97; p=0·020) among G6PD c.202T heterozygous girls but no evidence for protection among G6PD c.202T hemizygous boys and homozygous girls (OR 1·18, 0·99-1·40; p=0·056). Median follow-up for the mild disease cohort study was 2·24 years (IQR 2·22-2·85). G6PD c.202T had no effect on other common diseases of childhood in heterozygous girls (incidence rate ratio 0·98, 95% CI 0·86-1·11; p=0·82) or homozygous girls or hemizygous boys (0·93, 0·82-1·04; p=0·25), with the sole exception of a marginally significant increase in the incidence of helminth infections among heterozygous girls. INTERPRETATION: Heterozygous girls might be the driving force for the positive selection of G6PD deficiency alleles. Further studies are needed to definitively establish the mechanisms by which G6PD deficiency confers an advantage against malaria in heterozygous individuals. Such studies could lead to the development of new treatments. FUNDING: Wellcome Trust, UK Medical Research Council, European Union, and Foundation for the National Institutes of Health (as part of the Bill & Melinda Gates Grand Challenges in Global Health Initiative).