Canine junctional epidermolysis bullosa due to a novel mutation in LAMA3 with severe upper respiratory involvement.

BACKGROUND: Junctional epidermolysis bullosa (JEB) is a group of congenital blistering skin diseases characterized by clefting through the lamina lucida of the basement membrane zone. OBJECTIVES: To characterize the clinical and morphological features of a congenital mechanobullous disease in a litter of puppies with severe upper respiratory involvement, and to identify an associated genetic variant. ANIMALS: Five of eight puppies in an Australian cattle dog cross-bred litter showed signs of skin fragility. Three were stillborn and one died at one month of age. The two surviving puppies were presented with blistering skin disease and severe respiratory distress. Additionally, one unaffected sibling was examined and blood was obtained for genetic testing. METHODS AND MATERIALS: Post-mortem examination, histopathological evaluation and electron microscopy were performed. Whole genome sequencing (WGS) of one affected puppy was compared to a database of 522 dogs of 55 different breeds for variant analysis. Sanger sequencing of one additional affected and one unaffected sibling confirmed the variant. RESULTS: Clinically, severe mucocutaneous ulcers occurred in frictional areas with claw sloughing. Histopathological results revealed subepidermal clefts and electron microscopy confirmed the split in the lamina lucida. Post-mortem examination documented extensive pharyngeal and laryngeal lesions with granulation tissue and fibrinous exudate obscuring the airway. Moderate tracheal hypoplasia contributed. The WGS revealed a novel missense variant in the laminin α3-chain XP_537297.2p(Asp2867Val), with an autosomal recessive mode of inheritance. CONCLUSIONS AND CLINICAL RELEVANCE: A novel variant in LAMA3 caused a generalized and severe phenotype of JEB with an unique clinical presentation of upper airway obstruction.

Variants in CLCN1 and PDE4C Associated with Muscle Hypertrophy, Dysphagia, and Gait Abnormalities in Young French Bulldogs.

(1) Background: Muscle hypertrophy, swallowing disorders, and gait abnormalities are clinical signs common to many muscle diseases, including muscular dystrophies, non-dystrophic myotonias, genetic myopathies associated with deficiency of myostatin, and acquired inflammatory myopathies. Here, we investigated underlying causes of this triad of clinical signs in four young French bulldogs via muscle histopathology coupled with whole genome and Sanger sequencing. (2) Methods: Dogs were evaluated by veterinary clinical internists and neurologists, and biopsies were obtained for histopathological diagnosis. DNA was submitted for whole genome sequencing, followed by bioinformatics evaluation and confirmation of variants via Sanger sequencing in two cases. (3) Results: Two novel variants were identified. The first, found in two related French bulldogs, was a homozygous variant in the chloride channel gene CLCN1 known to cause non-dystrophic congenital myotonia, and the second, found in an unrelated French bulldog, was a heterozygous variant in the cAMP phosphodiesterase gene PDE4C, which is the major phosphodiesterase expressed in skeletal muscle and may play a role in decreasing muscle atrophy. An underlying molecular basis in one other case has not yet been identified. (4) Conclusions: Here, we identified two novel variants, one in the CLCN1 and one in the PDE4C gene, associated with clinical signs of muscle hypertrophy, dysphagia, and gait abnormalities, and we suggested other bases of these phenotypes in French bulldogs that are yet to be discovered. Identification of genes and deleterious variants associated with these clinical signs may assist breeders in improving the overall health of this very popular breed and may lead to the identification of new therapies to reverse muscle atrophy in people and animals with neuromuscular diseases.

Sequence Analysis of Six Candidate Genes in Miniature Schnauzers with Primary Hypertriglyceridemia.

Miniature Schnauzers are predisposed to primary hypertriglyceridemia (HTG). In this study, we performed whole genome sequencing (WGS) of eight Miniature Schnauzers with primary HTG and screened for risk variants in six HTG candidate genes: LPL, APOC2, APOA5, GPIHBP1, LMF1, and APOE. Variants were filtered to identify those present in ≥2 Miniature Schnauzers with primary HTG and uncommon (<10% allele frequency) in a WGS variant database including 613 dogs from 61 other breeds. Three variants passed filtering: an APOE TATA box deletion, an LMF1 intronic SNP, and a GPIHBP1 missense variant. The APOE and GPIHBP1 variants were genotyped in a cohort of 108 Miniature Schnauzers, including 68 with primary HTG and 40 controls. A multivariable regression model, including age and sex, did not identify an effect of APOE (estimate = 0.18, std. error = 0.14; p = 0.20) or GPIHBP1 genotypes (estimate = -0.26, std. error = 0.42; p = 0.54) on triglyceride concentration. In conclusion, we did not identify a monogenic cause for primary HTG in Miniature Schnauzers in the six genes evaluated. However, if HTG in Miniature Schnauzers is a complex disease resulting from the cumulative effects of multiple variants and environment, the identified variants cannot be ruled out as contributing factors.

A Novel CARMIL2 Immunodeficiency Identified in a Subset of Cavalier King Charles Spaniels with Pneumocystis and Bordetella Pneumonia.

Pet dogs are a valuable natural animal model for studying relationships between primary immunodeficiencies and susceptibility to Pneumocystis and other opportunistic respiratory pathogens. Certain breeds, such as the Cavalier King Charles Spaniel, are over-represented for Pneumocystis pneumonia (PCP), suggesting the presence of a primary immunodeficiency in the breed. Here, we report the discovery of a CARMIL2 nonsense variant in three Cavalier King Charles Spaniel dogs with either PCP (n = 2) or refractory Bordetella pneumonia (n = 1). CARMIL2 encodes a protein that plays critical roles in T-cell activation and other aspects of immune function. Deleterious CARMIL2 variants have recently been reported in human patients with PCP and other recurrent pneumonias. In addition to opportunistic respiratory infection, the affected dogs also exhibited other clinical manifestations of CARMIL2 deficiencies that have been reported in humans, including early-onset gastrointestinal disease, allergic skin disease, mucocutaneous lesions, abscesses, autoimmune disorders, and gastrointestinal parasitism. This discovery highlights the potential utility of a natural canine model in identifying and studying primary immunodeficiencies in patients affected by PCP.

Whole Animal Genome Sequencing: user-friendly, rapid, containerized pipelines for processing, variant discovery, and annotation of short-read whole genome sequencing data.

Advancements in massively parallel short-read sequencing technologies and the associated decreasing costs have led to large and diverse variant discovery efforts across species. However, processing high-throughput short-read sequencing data can be challenging with potential pitfalls and bioinformatics bottlenecks in generating reproducible results. Although a number of pipelines exist that address these challenges, these are often geared toward human or traditional model organism species and can be difficult to configure across institutions. Whole Animal Genome Sequencing (WAGS) is an open-source set of user-friendly, containerized pipelines designed to simplify the process of identifying germline short (SNP and indel) and structural variants (SVs) geared toward the veterinary community but adaptable to any species with a suitable reference genome. We present a description of the pipelines [adapted from the best practices of the Genome Analysis Toolkit (GATK)], along with benchmarking data from both the preprocessing and joint genotyping steps, consistent with a typical user workflow.

Current Classification of Canine Muscular Dystrophies and Identification of New Variants.

The spectrum of canine muscular dystrophies has rapidly grown with the recent identification of several more affected breeds and associated mutations. Defects include those in genes and protein products associated with the sarcolemma (dystrophin deficient X-linked muscular dystrophy and sarcoglycan-deficient limb-girdle muscular dystrophy) and with the extracellular matrix (collagen 6, laminin α2, and α-dystroglycan-deficient congenital muscular dystrophies). With the increasing application of whole genome sequencing and whole exome sequencing, the clinical and pathological spectra associated with specific neuromuscular genetic defects are constantly evolving. In this report, we provide a brief overview of the current status of gene defects reported in canine muscular dystrophies. We also report the causative mutations for novel forms of X-linked muscular dystrophy in Brittany spaniels and in a French bulldog.

Canine models of Charcot-Marie-Tooth: MTMR2, MPZ, and SH3TC2 variants in golden retrievers with congenital hypomyelinating polyneuropathy.

Congenital hypomyelinating polyneuropathy (HPN) restricted to the peripheral nervous system was reported in 1989 in two Golden Retriever (GR) littermates. Recently, four additional cases of congenital HPN in young, unrelated GRs were diagnosed via neurological examination, electrodiagnostic evaluation, and peripheral nerve pathology. Whole-genome sequencing was performed on all four GRs, and variants from each dog were compared to variants found across >1,000 other dogs, all presumably unaffected with HPN. Likely causative variants were identified for each HPN-affected GR. Two cases shared a homozygous splice donor site variant in MTMR2, with a stop codon introduced within six codons following the inclusion of the intron. One case had a heterozygous MPZ isoleucine to threonine substitution. The last case had a homozygous SH3TC2 nonsense variant predicted to truncate approximately one-half of the protein. Haplotype analysis using 524 GR established the novelty of the identified variants. Each variant occurs within genes that are associated with the human Charcot-Marie-Tooth (CMT) group of heterogeneous diseases, affecting the peripheral nervous system. Testing a large GR population (n = >200) did not identify any dogs with these variants. Although these variants are rare within the general GR population, breeders should be cautious to avoid propagating these alleles.

Novel COL6A3 frameshift variant in American Staffordshire Terrier dogs with Ullrich-like congenital muscular dystrophy.

Two (male and female) 10-month-old American Staffordshire Terrier littermates presented for progressive weakness, joint contracture, and distal limb joint hyperlaxity beginning around 6 months of age. Neurological examination, serum creatine kinase activity, infectious disease titers, cerebrospinal fluid analysis, and electrodiagnostic testing were performed. Muscle biopsies were collected for histopathology and immunofluorescence staining for localization of dystrophy associated proteins. Whole-genome sequencing (WGS) was performed on 1 affected dog. Variants were compared to a database of 671 unaffected dogs of multiple breeds. Histopathology confirmed a dystrophic phenotype and immunofluorescence staining of muscle cryosections revealed an absence of staining for collagen-6. WGS identified a homozygous 1 bp deletion in the COL6A3 gene, unique to the first affected dog. Sanger sequencing confirmed the homozygous presence of the frameshift variant in both affected dogs. This report describes the clinical features and most likely genetic basis of an Ullrich-like recessively inherited form of congenital muscular dystrophy in American Staffordshire Terriers.

Development and application of a next-generation sequencing protocol and bioinformatics pipeline for the comprehensive analysis of the canine immunoglobulin repertoire.

Profiling the adaptive immune repertoire using next generation sequencing (NGS) has become common in human medicine, showing promise in characterizing clonal expansion of B cell clones through analysis of B cell receptors (BCRs) in patients with lymphoid malignancies. In contrast, most work evaluating BCR repertoires in dogs has employed traditional PCR-based approaches analyzing the IGH locus only. The objectives of this study were to: (1) describe a novel NGS protocol to evaluate canine BCRs; (2) develop a bioinformatics pipeline for processing canine BCR sequencing data; and (3) apply these methods to derive insights into BCR repertoires of healthy dogs and dogs undergoing treatment for B-cell lymphoma. RNA from peripheral blood mononuclear cells of healthy dogs (n = 25) and dogs newly diagnosed with intermediate-to-large B-cell lymphoma (n = 18) with intent to pursue chemotherapy was isolated, converted into cDNA and sequenced by NGS. The BCR repertoires were identified and quantified using a novel analysis pipeline. The IGK repertoires of the healthy dogs were far less diverse compared to IGL which, as with IGH, was highly diverse. Strong biases at key positions within the CDR3 sequence were identified within the healthy dog BCR repertoire. For a subset of the dogs with B-cell lymphoma, clonal expansion of specific IGH sequences pre-treatment and reduction post-treatment was observed. The degree of expansion and reduction correlated with the clinical outcome in this subset. Future studies employing these techniques may improve disease monitoring, provide earlier recognition of disease progression, and ultimately lead to more targeted therapeutics.

Congenital muscular dystrophy in a dog with a LAMA2 gene deletion.

A 2-year-old female spayed dog was presented with a chronic history of short-strided gait and inability to completely open the jaw. Clinical signs were present since the dog was adopted from a humane society at a few months of age. Serum creatine kinase activity was abnormally high. Neurological examination, electromyography, muscle biopsies with immunofluorescent staining, and whole genome sequencing (WGS) were performed. A dystrophic phenotype was identified histologically in muscle biopsies, deficiency of laminin α2 protein was confirmed by immunofluorescent staining, and a deletion in the LAMA2 gene was identified by analysis of the WGS data. Congenital muscular dystrophy associated with a disease variant in LAMA2 was identified.

Tandem duplication within the DMD gene in Labrador retrievers with a mild clinical phenotype.

A form of dystrophinopathy with mild or subclinical neuromuscular signs has been previously reported in a family of Labrador retrievers. Markedly and persistently elevated creatine kinase activity was first noted at 6 months of age. Skeletal muscle biopsies revealed a dystrophic phenotype, with dystrophin non-detectable on western blotting and immunohistochemical staining, and with increased utrophin expression. In this report we demonstrate with western blotting that α-dystroglycan is present at essentially normal levels. Whole genome sequencing has also now revealed an approximately 400kb tandem genomic DNA duplication including exons 2-7 of the DMD gene that was inserted into intron 7 of the wild type gene. Skeletal muscle cDNA from 2 cases contained DMD transcripts as expected from an in-frame properly-spliced exon 2-7 tandem insertion. A similar 5' duplication involving DMD exons 2-7 has been reported in a human family with dilated cardiomyopathy but without skeletal myopathy. This is the 3rd confirmed mutation in the DMD gene in Labrador retrievers.

An EHPB1L1 Nonsense Mutation Associated with Congenital Dyserythropoietic Anemia and Polymyopathy in Labrador Retriever Littermates.

In this report, we describe a novel genetic basis for congenital dyserythropoietic anemia and polymyopathy in Labrador Retriever littermates characterized by incidental detection of marked microcytosis, inappropriate metarubricytosis, pelvic limb weakness and muscle atrophy. A similar syndrome has been described in English Springer Spaniel littermates with an early onset of anemia, megaesophagus, generalized muscle atrophy and cardiomyopathy. Muscle histopathology in both breeds showed distinctive pathological changes consistent with congenital polymyopathy. Using whole genome sequencing and mapping to the CanFam4 (Canis lupus familiaris reference assembly 4), a nonsense variant in the EHBP1L1 gene was identified in a homozygous form in the Labrador Retriever littermates. The mutation produces a premature stop codon that deletes approximately 90% of the protein. This variant was not present in the English Springer Spaniels. Currently, EHPB1L1 is described as critical to actin cytoskeletal organization and apical-directed transport in polarized epithelial cells, and through connections with Rab8 and a BIN1-dynamin complex generates membrane vesicles in the endocytic recycling compartment. Furthermore, EHBP1L1 knockout mice die early and develop severe anemia. The connection of EHBP1L1 to BIN1 and DMN2 functions is particularly interesting due to BIN1 and DMN2 mutations being causative in forms of centronuclear myopathy. This report, along with an independent study conducted by another group, are the first reports of an association of EHBP1L1 mutations with congenital dyserythropoietic anemia and polymyopathy.

Sarcoglycan A mutation in miniature dachshund dogs causes limb-girdle muscular dystrophy 2D.

BACKGROUND: A cohort of related miniature dachshund dogs with exercise intolerance, stiff gait, dysphagia, myoglobinuria, and markedly elevated serum creatine kinase activities were identified. METHODS: Muscle biopsy histopathology, immunofluorescence microscopy, and western blotting were combined to identify the specific pathologic phenotype of the myopathy, and whole genome SNP array genotype data and whole genome sequencing were combined to determine its genetic basis. RESULTS: Muscle biopsies were dystrophic. Sarcoglycanopathy, a form of limb-girdle muscular dystrophy, was suspected based on immunostaining and western blotting, where α, ß, and γ-sarcoglycan were all absent or reduced. Genetic mapping and whole genome sequencing identified a premature stop codon mutation in the sarcoglycan A subunit gene (SGCA). Affected dachshunds were confirmed on several continents. CONCLUSIONS: This first SGCA mutation found in dogs adds to the literature of genetic bases of canine muscular dystrophies and their usefulness as comparative models of human disease.

Muscular dystrophy-dystroglycanopathy in a family of Labrador retrievers with a LARGE1 mutation.

Alpha-dystroglycan (αDG) is a highly glycosylated cell surface protein with a significant role in cell-to-extracellular matrix interactions in muscle. αDG interaction with extracellular ligands relies on the activity of the LARGE1 glycosyltransferase that synthesizes and extends the heteropolysaccharide matriglycan. Abnormalities in αDG glycosylation and formation of matriglycan are the pathogenic mechanisms for the dystroglycanopathies, a group of congenital muscular dystrophies. Muscle biopsies were evaluated from related 6-week-old Labrador retriever puppies with poor suckling, small stature compared to normal litter mates, bow-legged stance and markedly elevated creatine kinase activities. A dystrophic phenotype with marked degeneration and regeneration, multifocal mononuclear cell infiltration and endomysial fibrosis was identified on muscle cryosections. Single nucleotide polymorphism (SNP) array genotyping data on the family members identified three regions of homozygosity in 4 cases relative to 8 controls. Analysis of whole genome sequence data from one of the cases identified a stop codon mutation in the LARGE1 gene that truncates 40% of the protein. Immunofluorescent staining and western blotting demonstrated the absence of matriglycan in skeletal muscle and heart from affected dogs. Compared to control, LARGE enzyme activity was not detected. This is the first report of a dystroglycanopathy in dogs.

Functional contexts of adipose and gluteal muscle tissue gene co-expression networks in the domestic horse.

A gene's response to an environment is tightly bound to the underlying genetic variation present in an individual's genome and varies greatly depending on the tissue it is being expressed in. Gene co-expression networks provide a mechanism to understand and interpret the collective transcriptional responses of genes. Here, we use the Camoco co-expression network framework to characterize the transcriptional landscape of adipose and gluteal muscle tissue in 83 domestic horses (Equus caballus) representing 5 different breeds. In each tissue, gene expression profiles, capturing transcriptional response due to variation across individuals, were used to build two separate, tissue-focused, genotypically-diverse gene co-expression networks. The aim of our study was to identify significantly co-expressed clusters of genes in each tissue, then compare the clusters across networks to quantify the extent that clusters were found in both networks as well as to identify clusters found in a single network. The known and unknown functions for each network were quantified using complementary, supervised and unsupervised approaches. First, supervised ontological enrichment was utilized to quantify biological functions represented by each network. Curated ontologies (GO and KEGG) were used to measure the known biological functions present in each tissue. Overall, a large percentage of terms (40.3% of GO and 41% of KEGG) were co-expressed in at least one tissue. Many terms were co-expressed in both tissues, however a small proportion of terms exhibited single tissue co-expression suggesting functional differentiation based on curated, functional annotation. To complement this, an unsupervised approach not relying on ontologies was employed. Strongly co-expressed sets of genes defined by Markov clustering identified sets of unannotated genes showing similar patterns of co-expression within a tissue. We compared gene sets across tissues and identified clusters of genes the either segregate in co-expression by tissue or exhibit high levels of co-expression in both tissues. Clusters were also integrated with GO and KEGG ontologies to identify gene sets containing previously curated annotations versus unannotated gene sets indicating potentially novel biological function. Coupling together these transcriptional datasets, we mapped the transcriptional landscape of muscle and adipose setting up a generalizable framework for interpreting gene function for additional tissues in the horse and other species.

Narrative review of therapies for chronic enteropathies in dogs and cats.

BACKGROUND: The optimal medical treatment for chronic enteropathy (CE) in dogs and cats is controversial. Sequential treatment using diet, antimicrobials, and immunosuppressive drugs is the most common strategy used by clinicians. OBJECTIVES: To review the evidence for the effectiveness of dietary, drug, and alternative health interventions for inducing clinical remission in dogs and cats with CE. ANIMALS: Retrospective study of dogs and cats with a diagnosis of chronic enteropathy. METHODS: MEDLINE and Centre for Agriculture and Bioscience International (CABI) databases (1950 to March 2017) were searched for randomized controlled trials (RCTs), observational studies, and case series. The primary outcome was induction of clinical remission. All studies were evaluated using the quality of evidence grading guidelines (I-IV), which assign a score defining the strength and quality of the evidence. RESULTS: Twenty-two studies (11 RCTs in dogs and 2 in cats and 9 cohort studies or case series) met the inclusion criteria for inducing remission of gastrointestinal (GI) signs. Of the 13 RCTs achieving grade I scores, 10 studies (totaling 218 dogs and 65 cats) compared single treatment: diet (n = 3), immunosuppressives (n = 3), antimicrobials (n = 2), anti-inflammatory drugs (n = 1), and probiotics (n = 1). Three case series (grade III) reported clinical remission using an elimination diet fed to 55 cats and use of enrofloxacin to induce remission in dogs with granulomatous colitis (2 studies totaling 16 dogs). CONCLUSIONS AND CLINICAL IMPORTANCE: The current evidence for treatment of CE is much greater in dogs than in cats. There is sufficient strong evidence to recommend the use of therapeutic GI diets, glucocorticoids, enrofloxacin, or some combination of these in dogs with CE. Therapeutic GI diets and glucocorticoids are most useful in cats with CE.

Reporting and analysis of repeated measurements in preclinical animals experiments.

A common feature of preclinical animal experiments is repeated measurement of the outcome, e.g., body weight measured in mice pups weekly for 20 weeks. Separate time point analysis or repeated measures analysis approaches can be used to analyze such data. Each approach requires assumptions about the underlying data and violations of these assumptions have implications for estimation of precision, and type I and type II error rates. Given the ethical responsibilities to maximize valid results obtained from animals used in research, our objective was to evaluate approaches to reporting repeated measures design used by investigators and to assess how assumptions about variation in the outcome over time impact type I and II error rates and precision of estimates. We assessed the reporting of repeated measures designs of 58 studies in preclinical animal experiments. We used simulation modelling to evaluate three approaches to statistical analysis of repeated measurement data. In particular, we assessed the impact of (a) repeated measure analysis assuming that the outcome had non-constant variation at all time points (heterogeneous variance) (b) repeated measure analysis assuming constant variation in the outcome (homogeneous variance), (c) separate ANOVA at individual time point in repeated measures designs. The evaluation of the three model fitting was based on comparing the p-values distributions, the type I and type II error rates and by implication, the shrinkage or inflation of standard error estimates from 1000 simulated dataset. Of 58 studies with repeated measures design, three provided a rationale for repeated measurement and 23 studies reported using a repeated-measures analysis approach. Of the 35 studies that did not use repeated-measures analysis, fourteen studies used only two time points to calculate weight change which potentially means collected data was not fully utilized. Other studies reported only select time points (n = 12) raising the issue of selective reporting. Simulation studies showed that an incorrect assumption about the variance structure resulted in modified error rates and precision estimates. The reporting of the validity of assumptions for repeated measurement data is very poor. The homogeneous variation assumption, which is often invalid for body weight measurements, should be confirmed prior to conducting the repeated-measures analysis using homogeneous covariance structure and adjusting the analysis using corrections or model specifications if this is not met.

A tightly regulated surface protein of Borrelia burgdorferi is not essential to the mouse-tick infectious cycle.

Borrelia burgdorferi synthesizes a variety of differentially regulated outer surface lipoproteins in the tick vector and in vertebrate hosts. Among these is OspD, a protein that is highly induced in vitro by conditions that mimic the tick environment. Using genetically engineered strains in which ospD is deleted, we demonstrate that this protein is not required for B. burgdorferi survival and infectivity in either the mouse or the tick. However, examination of both transcript levels and protein expression indicates that OspD expression is limited to a discrete window of time during B. burgdorferi replication within the tick. This time frame corresponds to tick detachment from the host following feeding, and expression of OspD continues during tick digestion of the blood meal but is low or undetectable after the tick has molted. The high level of OspD production correlates to the highest cell densities that B. burgdorferi is known to reach in vivo. Although OspD is nonessential to the infectious cycle of B. burgdorferi, the tight regulation of expression suggests a beneficial contribution of OspD to the spirochete during bacterial replication within the tick midgut.

The reporting characteristics of bovine respiratory disease clinical intervention trials published prior to and following publication of the REFLECT statement.

The goal of the REFLECT Statement (Reporting guidElines For randomized controLled trials in livEstoCk and food safeTy) (published in 2010) was to provide the veterinary research community with reporting guidelines tailored for randomized controlled trials for livestock and food safety. Our objective was to determine the prevalence of REFLECT Statement reporting of items 1-19 in controlled trials published in journals between 1970 and 2017 examining the comparative efficacy of FDA-registered antimicrobials against naturally acquired BRD (bovine respiratory disease) in weaned beef calves in Canada or the USA, and to compare the prevalence of reporting before and after 2010, when REFLECT was published. We divided REFLECT Statement, items 3, 5, 10, and 11 into subitems, because each dealt with multiple elements requiring separate assessment. As a result, 28 different items or subitems were evaluated independently. We searched MEDLINE® and CABI (CAB Abstracts® and Global Health®) (Web of Science™) in April 2017 and screened 2327 references. Two reviewers independently assessed the reporting of each item and subitem. Ninety-five references were eligible for the study. The reporting of the REFLECT items showed a point estimate for the prevalence ratio >1 (i.e. a higher proportion of studies published post-2010 reported this item compared to studies published pre-2010), apart from items 10.3, i.e., item 10, subitem 3 (who assigned study units to the interventions), 13 (the flow of study units through the study), 16 (number of study units in analysis), 18 (multiplicity), and 19 (adverse effects). Fifty-three (79%) of 67 studies published before 2010 and all 28 (100%) papers published after 2010 reported using a random allocation method in either the title, abstract, or methods (Prevalence ratio = 1.25; 95% CI (1.09,1.43)). However, 8 studies published prior to 2010 and 7 studies published post-2010 reported the term "systematic randomization" or variations of this term (which is not true randomization) to describe the allocation procedure. Fifty-five percent (37/67) of studies published pre-2010 reported blinding status (blinded/not blinded) of outcome assessors, compared to 24/28 (86%) of studies published post-2010 (Prevalence ratio = 1.5, 95% CI (1.19, 2.02)). The reporting of recommended items in journal articles in this body of work is generally improving; however, there is also evidence of confusion about what constitutes a random allocation procedure, and this suggests an educational need. As this study is observational, this precludes concluding that the publication of the REFLECT Statement was the cause of this trend.

Development and implementation of a novel immune thrombocytopenia bleeding score for dogs.

BACKGROUND: A method of quantifying clinical bleeding in dogs with immune thrombocytopenia (ITP) is needed because ITP patients have variable bleeding tendencies that inconsistently correlate with platelet count. A scoring system will facilitate patient comparisons and allow stratification based on bleeding severity in clinical trials. HYPOTHESIS/OBJECTIVES: To develop and evaluate a bleeding assessment tool for dogs, and a training course for improving its consistent implementation. ANIMALS: Client-owned dogs (n = 61) with platelet counts <50,000/µL; 34 classified as primary ITP, 17 as secondary ITP, and 10 as non-ITP. METHODS: A novel bleeding assessment tool, DOGiBAT, comprising bleeding grades from 0 (none) to 2 (severe) at 9 anatomic sites, was developed. Clinicians and technicians completed a training course and quiz before scoring thrombocytopenic patients. The training course was assessed by randomizing student volunteers to take the quiz with or without prior training. A logistic regression model assessed the association between training and quiz performance. The correlation of DOGiBAT score with platelet count and outcome measures was assessed in the thrombocytopenic dogs. RESULTS: Clinicians and technicians consistently applied the DOGiBAT, correctly scoring all quiz cases. The odds of trained students answering correctly were higher than those of untrained students (P < .0001). In clinical cases, DOGiBAT score and platelet count were inversely correlated (rs = -0.527, P < .0001), and DOGiBAT directly correlated with transfusion requirements (rs = 0.512, P < .0001) and hospitalization duration (rs = 0.35, P = .006). CONCLUSIONS AND CLINICAL IMPORTANCE: The DOGiBAT and assessment quiz are simple tools to standardize evaluation of bleeding severity. With further validation, the DOGiBAT may provide a clinically relevant metric to characterize ITP severity and monitor response in treatment trials.