Depicting deterministic variables within directed acyclic graphs (DAGs): An aid for identifying and interpreting causal effects involving derived variables and compositional data.

Deterministic variables are variables that are functionally determined by one or more parent variables. They commonly arise when a variable has been functionally created from one or more parent variables, as with derived variables, and in compositional data, where the 'whole' variable is determined from its 'parts'. This article introduces how deterministic variables may be depicted within directed acyclic graphs (DAGs) to help with identifying and interpreting causal effects involving derived variables and/or compositional data. We propose a two-step approach in which all variables are initially considered, and a choice is made whether to focus on the deterministic variable or its determining parents. Depicting deterministic variables within DAGs brings several benefits. It is easier to identify and avoid misinterpreting tautological associations, i.e., self-fulfilling associations between deterministic variables and their parents, or between sibling variables with shared parents. In compositional data, it is easier to understand the consequences of conditioning on the 'whole' variable, and correctly identify total and relative causal effects. For derived variables, it encourages greater consideration of the target estimand and greater scrutiny of the consistency and exchangeability assumptions. DAGs with deterministic variables are a useful aid for planning and interpreting analyses involving derived variables and/or compositional data.

Choice of Outcome in COVID-19 Studies and Implications for Policy: Mortality and Fatality.

In this brief communication, we discuss the confusion of mortality with fatality in the interpretation of evidence in the coronavirus disease 2019 (COVID-19) pandemic, and how this confusion affects the translation of science into policy and practice. We discuss how this confusion has influenced COVID-19 policy in France, Sweden, and the United Kingdom and discuss the implications for decision-making about COVID-19 vaccine distribution. We also discuss how this confusion is an example of a more general statistical fallacy we term the "Missing Link Fallacy."

Causal and Associational Language in Observational Health Research: A Systematic Evaluation.

We estimated the degree to which language used in the high-profile medical/public health/epidemiology literature implied causality using language linking exposures to outcomes and action recommendations; examined disconnects between language and recommendations; identified the most common linking phrases; and estimated how strongly linking phrases imply causality. We searched for and screened 1,170 articles from 18 high-profile journals (65 per journal) published from 2010-2019. Based on written framing and systematic guidance, 3 reviewers rated the degree of causality implied in abstracts and full text for exposure/outcome linking language and action recommendations. Reviewers rated the causal implication of exposure/outcome linking language as none (no causal implication) in 13.8%, weak in 34.2%, moderate in 33.2%, and strong in 18.7% of abstracts. The implied causality of action recommendations was higher than the implied causality of linking sentences for 44.5% or commensurate for 40.3% of articles. The most common linking word in abstracts was "associate" (45.7%). Reviewers' ratings of linking word roots were highly heterogeneous; over half of reviewers rated "association" as having at least some causal implication. This research undercuts the assumption that avoiding "causal" words leads to clarity of interpretation in medical research.

The Association Between Childhood Leukemia and Population Mixing: An Artifact of Focusing on Clusters?

BACKGROUND: Studies investigating the population-mixing hypothesis in childhood leukemia principally use two analytical approaches: (1) nonrandom selection of areas according to specific characteristics, followed by comparisons of their incidence of childhood leukemia with that expected based on the national average; and (2) regression analyses of region-wide data to identify characteristics associated with the incidence of childhood leukemia. These approaches have generated contradictory results. We compare these approaches using observed and simulated data. METHODS: We generated 10,000 simulated regions using the correlation structure and distributions from a United Kingdom dataset. We simulated cases using a Poisson distribution with the incidence rate set to the national average assuming the null hypothesis that only population size drives the number of cases. Selection of areas within each simulated region was based on characteristics considered responsible for elevated infection rates (population density and inward migration) and/or elevated leukemia rates. We calculated effect estimates for 10,000 simulations and compared results to corresponding observed data analyses. RESULTS: When the selection of areas for analysis is based on apparent clusters of childhood leukemia, biased assessments occur; the estimated 5-year incidence of childhood leukemia ranged between zero and eight per 10,000 children in contrast to the simulated two cases per 10,000 children, similar to the observed data. Performing analyses on region-wide data avoids these biases. CONCLUSIONS: Studies using nonrandom selection to investigate the association between childhood leukemia and population mixing are likely to have generated biased findings. Future studies can avoid such bias using a region-wide analytical strategy. See video abstract at, http://links.lww.com/EDE/B431.

Doppler ultrasound can be used to monitor umbilical arterial blood flow in lightly sedated pigs at multiple gestational ages.

Doppler ultrasound was performed under moderate sedation (ketamine and azaperone) for 30min to monitor umbilical arterial (UA) blood flow in one uterine horn of Large White×Landrace gilts (n=23) at Gestational Days (GD) 30, 45, 60 and 90. Gilts were scanned before they were killed to examine relationships between litter size, sex ratio and five UA parameters (peak systolic velocity (PSV), end diastolic velocity (EDV), A/B (PSV to EDV) ratio, fetal heart rate (FHR) and resistance index (RI)). In gilts in which scans were obtained from all fetuses in the scanned horn, relationships between UA parameters, and fetal weight and sex were examined. A subset of gilts were sedated, scanned and recovered (SSR) earlier in gestation (GD30 or GD45) to assess the effects of sedation on later fetal development by comparison with control litters (no previous sedation). Temporal changes were observed in all UA parameters (P≤0.001). At GD60 and GD90, FHR decreased with increasing duration of sedation (P≤0.001). Sex ratio and fetal weight affected UA blood flow, whereas litter size and fetal sex did not. SSR at GD30 and GD45 was associated with decreased fetal weight at GD60 (P≤0.001) and GD90 (P=0.06) respectively, compared with controls. These results suggest maternal sedation during gestation affects fetal development, which should be investigated further. Measuring UA blood flow in growth-restricted porcine fetuses throughout gestation may be feasible.

Risk estimates of recurrent congenital anomalies in the UK: a population-based register study.

BACKGROUND: Recurrence risks for familial congenital anomalies in successive pregnancies are known, but this information for major structural anomalies is lacking. We estimated the absolute and relative risks of recurrent congenital anomaly in the second pregnancy for women with a history of a congenital anomaly in the first pregnancy, for all major anomaly groups and subtypes. METHODS: Population-based register data on 18,605 singleton pregnancies affected by major congenital anomaly occurring in 872,493 singleton stillbirths, live births and terminations of pregnancy for fetal anomaly were obtained from the Northern Congenital Abnormality Survey, North of England, UK, for 1985-2010. Absolute risks (ARs) and relative risks (RRs) for recurrent congenital anomaly (overall, from a similar group, from a dissimilar group) in the second pregnancy were estimated by history of congenital anomaly (overall, by group, by subtype) in the first pregnancy. RESULTS: The estimated prevalences of congenital anomaly in first and second pregnancies were 275 (95% CI 270-281) and 163 (95% CI 159-168) per 10,000 respectively. For women whose first pregnancy was affected by congenital anomaly, the AR of recurrent congenital anomaly in the second pregnancy was 408 (95% CI 365-456) per 10,000, 2.5 (95% CI 2.3-2.8, P < 0.0001) times higher than for those with unaffected first pregnancies. For similar anomalies, the recurrence risk was considerably elevated (RR = 23.8, 95% CI 19.6-27.9, P < 0.0001), while for dissimilar anomalies the increase was more modest (RR = 1.4, 95% CI 1.2-1.6, P = 0.001), although the ARs for both were 2%. CONCLUSIONS: Absolute recurrence risks varied between 1 in 20 and 1 in 30 for most major anomaly groups. At pre-conception and antenatal counselling, women whose first pregnancy was affected by a congenital anomaly and who are planning a further pregnancy may find it reassuring that, despite high relative risks, the absolute recurrence risk is relatively low.

Microbiota in Exhaled Breath Condensate and the Lung.

The lung microbiota is commonly sampled using relatively invasive bronchoscopic procedures. Exhaled breath condensate (EBC) collection potentially offers a less invasive alternative for lung microbiota sampling. We compared lung microbiota samples retrieved by protected specimen brushings (PSB) and exhaled breath condensate collection. We also sought to assess whether aerosolized antibiotic treatment would influence the lung microbiota and whether this change could be detected in EBC. EBC was collected from 6 conscious sheep and then from the same anesthetized sheep during mechanical ventilation. Following the latter EBC collection, PSB samples were collected from separate sites within each sheep lung. On the subsequent day, each sheep was then treated with nebulized colistimethate sodium. Two days after nebulization, EBC and PSB samples were again collected. Bacterial DNA was quantified using 16S rRNA gene quantitative PCR. The V2-V3 region of the 16S rRNA gene was amplified by PCR and sequenced using Illumina MiSeq. Quality control and operational taxonomic unit (OTU) clustering were performed with mothur. The EBC samples contained significantly less bacterial DNA than the PSB samples. The EBC samples from anesthetized animals clustered separately by their bacterial community compositions in comparison to the PSB samples, and 37 bacterial OTUs were identified as differentially abundant between the two sample types. Despite only low concentrations of colistin being detected in bronchoalveolar lavage fluid, PSB samples were found to differ by their bacterial compositions before and after colistimethate sodium treatment. Our findings indicate that microbiota in EBC samples and PSB samples are not equivalent.IMPORTANCE Sampling of the lung microbiota usually necessitates performing bronchoscopic procedures that involve a hospital visit for human participants and the use of trained staff. The inconvenience and perceived discomfort of participating in this kind of research may deter healthy volunteers and may not be a safe option for patients with advanced lung disease. This study set out to evaluate a less invasive method for collecting lung microbiota samples by comparing samples taken via protected specimen brushings (PSB) to those taken via exhaled breath condensate (EBC) collection. We found that there was less bacterial DNA in EBC samples compared with that in PSB samples and that there were differences between the bacterial communities in the two sample types. We conclude that while EBC and PSB samples do not produce equivalent microbiota samples, the study of the EBC microbiota may still be of interest.

Variability of the Sheep Lung Microbiota.

UNLABELLED: Sequencing technologies have recently facilitated the characterization of bacterial communities present in lungs during health and disease. However, there is currently a dearth of information concerning the variability of such data in health both between and within subjects. This study seeks to examine such variability using healthy adult sheep as our model system. Protected specimen brush samples were collected from three spatially disparate segmental bronchi of six adult sheep (age, 20 months) on three occasions (day 0, 1 month, and 3 months). To further explore the spatial variability of the microbiotas, more-extensive brushing samples (n = 16) and a throat swab were taken from a separate sheep. The V2 and V3 hypervariable regions of the bacterial 16S rRNA genes were amplified and sequenced via Illumina MiSeq. DNA sequences were analyzed using the mothur software package. Quantitative PCR was performed to quantify total bacterial DNA. Some sheep lungs contained dramatically different bacterial communities at different sampling sites, whereas in others, airway microbiotas appeared similar across the lung. In our spatial variability study, we observed clustering related to the depth within the lung from which samples were taken. Lung depth refers to increasing distance from the glottis, progressing in a caudal direction. We conclude that both host influence and local factors have impacts on the composition of the sheep lung microbiota. IMPORTANCE: Until recently, it was assumed that the lungs were a sterile environment which was colonized by microbes only during disease. However, recent studies using sequencing technologies have found that there is a small population of bacteria which exists in the lung during health, referred to as the "lung microbiota." In this study, we characterize the variability of the lung microbiotas of healthy sheep. Sheep not only are economically important animals but also are often used as large animal models of human respiratory disease. We conclude that, while host influence does play a role in dictating the types of microbes which colonize the airways, it is clear that local factors also play an important role in this regard. Understanding the nature and influence of these factors will be key to understanding the variability in, and functional relevance of, the lung microbiota.

Partial deletion of chromosome 8 ß-defensin cluster confers sperm dysfunction and infertility in male mice.

ß-defensin peptides are a family of antimicrobial peptides present at mucosal surfaces, with the main site of expression under normal conditions in the male reproductive tract. Although they kill microbes in vitro and interact with immune cells, the precise role of these genes in vivo remains uncertain. We show here that homozygous deletion of a cluster of nine ß-defensin genes (DefbΔ9) in the mouse results in male sterility. The sperm derived from the mutants have reduced motility and increased fragility. Epididymal sperm isolated from the cauda should require capacitation to induce the acrosome reaction but sperm from the mutants demonstrate precocious capacitation and increased spontaneous acrosome reaction compared to wild-types but have reduced ability to bind the zona pellucida of oocytes. Ultrastructural examination reveals a defect in microtubule structure of the axoneme with increased disintegration in mutant derived sperm present in the epididymis cauda region, but not in caput region or testes. Consistent with premature acrosome reaction, sperm from mutant animals have significantly increased intracellular calcium content. Thus we demonstrate in vivo that ß-defensins are essential for successful sperm maturation, and their disruption leads to alteration in intracellular calcium, inappropriate spontaneous acrosome reaction and profound male infertility.

Langerhans cell histiocytosis in children with congenital anomalies: a population-based record linkage study.

BACKGROUND: The etiology of Langerhans cell histiocytosis (LCH), a rare cancer-like disorder of the immune system, is largely unknown although a genetic component has been suggested based on familial cases, and reports of chromosome instability and genetic mutation. Associations between various cancers and congenital anomalies have been reported and although congenital anomalies have been noted in children with LCH only one study to date has reported their frequency. An association between congenital anomalies and LCH may suggest a common etiological pathway, in particular, a genetic pathway. METHODS: Data from two coterminous registries in the same geographic region were used. All cases of LCH on the Northern Region Young Persons Malignant Disease Register diagnosed between 1985 and 2010 were cross-matched with live-born cases of congenital anomaly registered by the Northern Congenital Abnormality Survey. RESULTS: A total of 819,890 children and young people were born during 1985 to 2008. Of these, 13,799 (1.7%) had a congenital anomaly and 39 (0.005%) were diagnosed with LCH. Three LCH cases were identified among those with congenital anomalies, all three of whom had congenital heart disease. The relative risk of LCH for those with a congenital anomaly, compared with those without, was 4.87 (95% confidence interval, 1.50-15.81; p = 0.03). CONCLUSION: LCH was associated with congenital anomaly in a small but statistically significant number of patients, raising the possibility of a common genetic pathway in some cases.

Variation in health care for children and young people with cerebral palsies: a retrospective multicentre audit study.

AIM: To explore the provision and variations in care for children and young people with cerebral palsies (CP) registered with the population-based North of England Collaborative Cerebral Palsy Survey (NECCPS). METHOD: This is a retrospective multicentre record audit of 389 children with CP (220 males, 148 females, 21 no data; median age at time of audit 12y 3mo), born between 1995 and 2002. Data were collected on cranial magnetic resonance imaging (MRI), hip and spine surveillance and management, and pain presence and management. Variations over time and between the districts in the north of England (Northumberland, North and West Cumbria, North and South Tyneside, Newcastle-upon-Tyne, Gateshead, Sunderland, Durham, Darlington, Bishop Auckland, Hartlepool, Stockton-on-Tees, Middlesbrough, Redcar, and Cleveland), and by socio-economic status (SES) (estimated from the Index of Multiple Deprivation [IMD] 2004) were estimated by generalized estimating equations. RESULTS: There was significant variation between districts in access to MRI (p<0.001), orthopaedic surgeons (p=0.005), recording state of spine (p<0.001), and discussions about pain (p<0.001). Fifty-seven per cent (95% CI 52-62) had evidence of a reported MRI brain scan, the proportion of which increased over time (p<0.001). Sixty-seven per cent (95% CI 62-71) had a discussion about pain recorded. Of those in pain, 87% (95% CI 80-93) had a pain management plan. The proportion with documented discussion about pain increased with increasing SES (p=0.04). INTERPRETATION: The provision of care for children with CP in the north of England varies between districts. Internationally agreed, evidence-based standards are urgently needed to ensure more equitable health care and improved outcomes for all.

Pre-existing diabetes, maternal glycated haemoglobin, and the risks of fetal and infant death: a population-based study.

AIMS/HYPOTHESIS: Pre-existing diabetes is associated with an increased risk of stillbirth, but few studies have excluded the effect of congenital anomalies. This study used data from a long-standing population-based survey of women with pre-existing diabetes to investigate the risks of fetal and infant death and quantify the contribution of glycaemic control. METHODS: All normally formed singleton offspring of women with pre-existing diabetes (1,206 with type 1 diabetes and 342 with type 2 diabetes) in the North of England during 1996-2008 were identified from the Northern Diabetes in Pregnancy Survey. RRs of fetal death (≥20 weeks of gestation) and infant death were estimated by comparison with population data from the Northern Perinatal Morbidity and Mortality Survey. Predictors of fetal and infant death in women with pre-existing diabetes were examined by logistic regression. RESULTS: The prevalence of fetal death in women with diabetes was over four times greater than in those without (RR 4.56 [95% CI 3.42, 6.07], p < 0.0001), and for infant death it was nearly doubled (RR 1.86 [95% CI 1.00, 3.46], p = 0.046). There was no difference in the prevalence of fetal death (p = 0.51) or infant death (p = 0.70) between women with type 1 diabetes and women with type 2 diabetes. There was no evidence that the RR of fetal and infant death had changed over time (p = 0.95). Increasing periconception HbA1c concentration above 49 mmol/mol (6.6%) (adjusted odds ratio [aOR] 1.02 [95% CI 1.00, 1.04], p = 0.01), prepregnancy retinopathy (aOR 2.05 [95% CI 1.04, 4.05], p = 0.04) and lack of prepregnancy folic acid consumption (aOR 2.52 [95% CI 1.12, 5.65], p = 0.03) were all independently associated with increased odds of fetal and infant death. CONCLUSIONS/INTERPRETATION: Pre-existing diabetes is associated with a substantially increased risk of fetal and infant death in normally formed offspring, the effect of which is largely moderated by glycaemic control.

How to develop causal directed acyclic graphs for observational health research: a scoping review.

Causal directed acyclic graphs (DAGs) serve as intuitive tools to visually represent causal relationships between variables. While they find widespread use in guiding study design, data collection and statistical analysis, their adoption remains relatively rare in the domain of psychology. In this paper we describe the relevance of DAGs for health psychology, review guidelines for developing causal DAGs, and offer recommendations for their development. A scoping review searching for papers and resources describing guidelines for DAG development was conducted. Information extracted from the eligible papers and resources (n = 11) was categorised, and results were used to formulate recommendations. Most records focused on DAG development for data analysis, with similar steps outlined. However, we found notable variations on how to implement confounding variables (i.e., sequential inclusion versus exclusion). Also, how domain knowledge should be integrated in the development process was scarcely addressed. Only one paper described how to perform a literature search for DAG development. Key recommendations for causal DAG development are provided and discussed using an illustrative example.

Consistent detection of Trypanosoma brucei but not T. congolense DNA in faeces of experimentally infected cattle.

Animal African trypanosomiasis (AAT) is a significant food security and economic burden in sub-Saharan Africa. Current AAT empirical and immunodiagnostic surveillance tools suffer from poor sensitivity and specificity, with blood sampling requiring animal restraint and trained personnel. Faecal sampling could increase sampling accessibility, scale, and species range. Therefore, this study assessed feasibility of detecting Trypanosoma DNA in the faeces of experimentally-infected cattle. Holstein-Friesian calves were inoculated with Trypanosoma brucei brucei AnTat 1.1 (n = 5) or T. congolense Savannah IL3000 (n = 6) in separate studies. Faecal and blood samples were collected concurrently over 10 weeks and screened using species-specific PCR and qPCR assays. T. brucei DNA was detected in 85% of post-inoculation (PI) faecal samples (n = 114/134) by qPCR and 50% by PCR between 4 and 66 days PI. However, T. congolense DNA was detected in just 3.4% (n = 5/145) of PI faecal samples by qPCR, and none by PCR. These results confirm the ability to consistently detect T. brucei DNA, but not T. congolense DNA, in infected cattle faeces. This disparity may derive from the differences in Trypanosoma species tissue distribution and/or extravasation. Therefore, whilst faeces are a promising substrate to screen for T. brucei infection, blood sampling is required to detect T. congolense in cattle.

Ciliary tip actin dynamics regulate photoreceptor outer segment integrity.

As signalling organelles, cilia regulate their G protein-coupled receptor content by ectocytosis, a process requiring localised actin dynamics to alter membrane shape. Photoreceptor outer segments comprise an expanse of folded membranes (discs) at the tip of highly-specialised connecting cilia, into which photosensitive GPCRs are concentrated. Discs are shed and remade daily. Defects in this process, due to mutations, cause retinitis pigmentosa (RP). Whilst fundamental for vision, the mechanism of photoreceptor disc generation is poorly understood. Here, we show membrane deformation required for disc genesis is driven by dynamic actin changes in a process akin to ectocytosis. We show RPGR, a leading RP gene, regulates actin-binding protein activity central to this process. Actin dynamics, required for disc formation, are perturbed in Rpgr mouse models, leading to aborted membrane shedding as ectosome-like vesicles, photoreceptor death and visual loss. Actin manipulation partially rescues this, suggesting the pathway could be targeted therapeutically. These findings help define how actin-mediated dynamics control outer segment turnover.

Tracking Ovine Pulmonary Adenocarcinoma Development Using an Experimental Jaagsiekte Sheep Retrovirus Infection Model.

Ovine pulmonary adenocarcinoma (OPA) is an infectious, neoplastic lung disease of sheep that causes significant animal welfare and economic issues throughout the world. Understanding OPA pathogenesis is key to developing tools to control its impact. Central to this need is the availability of model systems that can monitor and track events after Jaagsiekte sheep retrovirus (JSRV) infection. Here, we report the development of an experimentally induced OPA model intended for this purpose. Using three different viral dose groups (low, intermediate and high), localised OPA tumour development was induced by bronchoscopic JSRV instillation into the segmental bronchus of the right cardiac lung lobe. Pre-clinical OPA diagnosis and tumour progression were monitored by monthly computed tomography (CT) imaging and trans-thoracic ultrasound scanning. Post mortem examination and immunohistochemistry confirmed OPA development in 89% of the JSRV-instilled animals. All three viral doses produced a range of OPA lesion types, including microscopic disease and gross tumours; however, larger lesions were more frequently identified in the low and intermediate viral groups. Overall, 31% of JSRV-infected sheep developed localised advanced lesions. Of the sheep that developed localised advanced lesions, tumour volume doubling times (calculated using thoracic CT 3D reconstructions) were 14.8 ± 2.1 days. The ability of ultrasound to track tumour development was compared against CT; the results indicated a strong significant association between paired CT and ultrasound measurements at each time point (R2 = 0.799, p < 0.0001). We believe that the range of OPA lesion types induced by this model replicates aspects of naturally occurring disease and will improve OPA research by providing novel insights into JSRV infectivity and OPA disease progression.