Short course of immune-suppressive doses of prednisolone, evaluated through a prospective double-masked placebo-controlled clinical trial in healthy Beagles, is associated with sustained modifications in renal, hydration, and electrolytic status.

OBJECTIVE: To investigate the effects and duration of orally administered prednisolone on renal function evaluated by glomerular filtration rate (GFR) determination and creatinine (Cr) and symmetric dimethylarginine (SDMA) concentrations as well as on urinalysis, electrolytes, and hydric status in healthy dogs. ANIMALS: 14 healthy Beagles. PROCEDURES: In this prospective double-masked placebo-controlled study, dogs were randomized after baseline evaluation to receive a 7-day course of either prednisolone (1.5 to 2.0 mg/kg, PO, q 12 h) or a placebo. A repeated-measure design was performed, each dog participating in 4 successive sampling sessions. Clinical data, systolic blood pressure, CBC, and biochemical analyses including serum SDMA concentration, GFR determination, urine output quantification, and complete urinalysis were performed for all dogs the day before (D0) and at the end of steroid administration (D7) as well as 2 weeks (D21) and 4 weeks (D35) after the end of treatment. RESULTS: At D7, when compared with baseline, GFR increased significantly in treated dogs, whereas creatinine and SDMA concentrations decreased significantly. GFR and Cr but not SDMA modifications persisted significantly at D21. None of the variables differed significantly from baseline at D35. The OR of presenting an albumin band on urine electrophoresis was 2.4 times as high in treated versus control dogs (OR, 36; 95% CI, 1.8 to 719.4; P = 0.02). CLINICAL RELEVANCE: A short-term course of immune-suppressive prednisolone treatment in healthy dogs leads to a sustained but reversible renal hyperfiltration state. Modification in electrolytic variables can affect the clinical interpretation of blood work in such patients.

Hydroxychloroquine lung pharmacokinetics in critically ill patients with COVID-19.

Different dosage regimens of hydroxychloroquine (HCQ) have been used to manage COVID-19 (coronavirus disease 2019) patients, with no information on lung exposure in this population. The aim of our study was to evaluate HCQ concentrations in the lung epithelial lining fluid (ELF) in patients infected with SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), the virus that causes COVID-19. This was a retrospective, observational, multicentre, pharmacokinetic study of HCQ in critically ill COVID-19 patients. No additional interventions or additional samples compared with standard care of these patients were conducted in our teaching hospital. We included all intubated COVID-19 patients treated with crushed HCQ tablets, regardless of the dosage administered by nasogastric tube. Blood and bronchoalveolar lavage samples (n = 28) were collected from 22 COVID-19 patients and total HCQ concentrations in ELF were estimated. Median (interquartile range) HCQ plasma concentrations were 0.09 (0.06-0.14) mg/L and 0.07 (0.05-0.08) mg/L for 400 mg × 1/day and 200 mg × 3/day, respectively. Median HCQ ELF concentrations were 3.74 (1.10-7.26) mg/L and 1.81 (1.20-7.25) for 400 mg × 1/day and 200 mg × 3/day, respectively. The median ratio of ELF/plasma concentrations was 40.0 (7.3-162.7) and 21.2 (18.4-109.5) for 400 mg × 1/day and 200 mg × 3/day, respectively. ELF exposure is likely to be underestimated from HCQ concentrations in plasma. In clinical practice, low plasma concentrations should not induce an increase in drug dosage because lung exposure may already be high.

The use of thermometer protective sheets provides reliable measurement of rectal temperature: a prospective study in 500 dogs.

OBJECTIVES: To measure and compare rectal temperature measurement with and without thermometer protective sheaths. To survey veterinary practices regarding thermometer use and disinfection. MATERIALS AND METHODS: Thermometers were validated with and without protective sheaths for accuracy and repeatability in a water bath with a high precision thermometer as reference. Then, the rectal temperature of 500 dogs was measured with and without protective sheaths in randomised order. The difference in temperature measured by the two methods was analysed with a generalised linear model with order of temperature measurement, life stage, dog size, body condition, level of consciousness, class of temperature and operator as explanatory variables. Practical thermometer use and disinfection was surveyed by online questionnaire. RESULTS: In the pre-clinical phase, study thermometers recorded average temperature differences of −0.05 ± 0.07°C (mean ± SD) and −0.03 ± 0.08°C with and without sheath, respectively and the coefficients of variation ranged from 0.1 to 0.5% with and without sheath. In the clinical study, temperatures measured with and without sheaths differed by less than ±0.2°C for 92% of the measurements. None of the explanatory variables were associated with the observed differences. Twenty-five percent of survey respondents were aware of the guidelines for disinfection of thermometers. CLINICAL SIGNIFICANCE: Thermometer protective sheaths do not affect the measurement of canine rectal temperature in the clinic.

Mathematical modeling and simulation in animal health. Part III: Using nonlinear mixed-effects to characterize and quantify variability in drug pharmacokinetics.

A common feature of human and veterinary pharmacokinetics is the importance of identifying and quantifying the key determinants of between-patient variability in drug disposition and effects. Some of these attributes are already well known to the field of human pharmacology such as bodyweight, age, or sex, while others are more specific to veterinary medicine, such as species, breed, and social behavior. Identification of these attributes has the potential to allow a better and more tailored use of therapeutic drugs both in companion and food-producing animals. Nonlinear mixed effects (NLME) have been purposely designed to characterize the sources of variability in drug disposition and response. The NLME approach can be used to explore the impact of population-associated variables on the relationship between drug administration, systemic exposure, and the levels of drug residues in tissues. The latter, while different from the method used by the US Food and Drug Administration for setting official withdrawal times (WT) can also be beneficial for estimating WT of approved animal drug products when used in an extralabel manner. Finally, NLME can also prove useful to optimize dosing schedules, or to analyze sparse data collected in situations where intensive blood collection is technically challenging, as in small animal species presenting limited blood volume such as poultry and fish.

Comparison of routinely used intestinal biopsy forceps in dogs: an ex vivo histopathological approach.

OBJECTIVES: To compare the quality of duodenal and ileal samples obtained with different biopsy forceps. METHODS: Fifteen dogs were included in a prospective ex vivo study. After euthanasia, the duodenum and the ileum were sampled with four different forceps and evaluated according to a standardised scoring system. The biopsy forceps evaluated had alligator jaws or cups with smooth edge with or without a needle. RESULTS: The global quality of the biopsies was better in the ileum that in the duodenum regardless of the biopsy forceps. Biopsy forceps with smooth edge including a needle resulted in fewer artefacts than biopsy forceps with smooth edge but no needle in both sites and those with alligator jaws without a needle provided deeper biopsies than those with smooth edge without a needle only in the duodenum. There was no effect of the biopsy forceps type on the size of the biopsies. CLINICAL SIGNIFICANCE: Our findings may aid in choosing the appropriate type of forceps for intestinal biopsy.

Plasma exogenous creatinine clearance in clinically healthy cats: comparison with urinary exogenous creatinine clearance, tentative reference intervals and indexation to bodyweight.

Glomerular filtration rate (GFR) is considered to be the best indicator of overall kidney function. The major objectives of this study were to compare plasma exogenous creatinine clearance (PECC) with a reference method, to establish reference intervals (RIs) for PECC and to assess the effects of indexation of GFR to bodyweight (BW) in cats. PECC was compared with urinary clearance of exogenous creatinine (UECC) in six clinically healthy domestic shorthair cats (experiment 1). Tentative RIs were determined according to current guidelines and the effects of indexation to BW and of covariables on GFR were assessed in 43 clinically healthy cats of various breeds (experiment 2). PECC was 15% higher than UECC (P <0.01), but the two estimates were strongly correlated (r(2)=0.97, P = 0.001). RIs for PECC were 6.4-21.3 mL/min or 1.2-4.9 mL/min/kg. The absolute (i.e. non-indexed) GFR value was not dependent on BW. Thus, indexation of GFR to BW in cats would not standardize the GFR value, but could introduce bias in clinical interpretation. Significant effects of breed, plasma protein concentration and plasma albumin concentration on GFR were demonstrated. Plasma concentrations of urea and creatinine, when assessed separately, were also weakly correlated with GFR in healthy cats. These combined findings contribute to a better understanding of renal function assessment in cats.

Individual prediction regions for multivariate longitudinal data with small samples.

Follow-up is more and more used in medicine/doping control to identify abnormal results in an individual. Currently, follow-ups are mostly carried out variable by variable using "reference intervals" that contain the values observable in 100(1-α)% of healthy/undoped individuals. Observations of the evolution of the variables over time in a sample of N healthy/undoped individuals, allows these reference intervals to be individualized by taking into account the possible effect of covariables and some previous observations of these variables obtained when the individual was healthy/undoped. For each variable these individualized intervals should contain 100(1-α)% of observable values compatible with previous observed values in this individual. General methods to build these intervals are available, but they allow only a variable by variable follow-up whatever the possible correlations over time between them. In this article, we propose a general method to jointly follow-up several correlated variables over time. This methodology relies on a multivariate linear mixed effects model. We first provide a method to estimate the model's parameters. In an asymptotic framework (N large enough), we then derive a (1-α) individualized prediction region. Sometimes, the sample size N is not large enough for the asymptotic framework to give a reasonable prediction region. It is for this reason, we propose and compare three different prediction regions that should behave better for small N. Finally, the whole methodology is illustrated by the follow-up of kidney insufficiency in cats.

Confidence intervals of reference limits in small reference sample groups.

BACKGROUND: Reference intervals are the most common tool used to interpret results of laboratory tests. However, in veterinary clinical pathology, the number of available reference individuals is often small. OBJECTIVES: The purpose of this study was to investigate the effects of small reference sample groups on the imprecision of the reference limits. METHODS: Gaussian and log-Gaussian distributions of 10 ≤ n ≤ 750 values were analyzed. Reference limits and 90% confidence interval of limits (90% CI) were calculated. Imprecision of limits was estimated by the ratio of the width of the 90% CI: width of the reference interval (WCI/WRI). RESULTS: For Gaussian distributions, the WCI/WRI ratio cannot be expected to be lower than 0.2 when n < 55. In log-Gaussian distributions, the ratio greatly increases for the upper limit with skewness toward high values, whereas it moderately decreases for the lower limit. CONCLUSION: Independent of the size of the reference sample group, it is very important to report the CIs of the reference limits, which can be very large for small reference sample groups. When the sample size is very small (n < 20), calculations maybe misleading and it is better to instead report all values.

Safety of spironolactone in dogs with chronic heart failure because of degenerative valvular disease: a population-based, longitudinal study.

BACKGROUND: Spironolactone treatment in humans is associated with an increased risk of hyperkalemia and renal dysfunction. HYPOTHESIS: Dogs with cardiac disease treated with spironolactone, in addition to conventional therapy, are not at higher risk for adverse events (AEs) than those receiving solely conventional therapy. ANIMALS: One hundred and ninety-six client-owned dogs with naturally occurring myxomatous mitral valve disease. METHODS: Prospective, double-blinded field study with dogs randomized to receive either spironolactone (2 mg/kg once a day) or placebo in addition to conventional therapy (angiotensin-converting enzyme inhibitor, plus furosemide and digoxin if needed). Safety was compared between treatment groups, using the frequency of AEs, death caused by cardiac disease, renal disease, or both, and variations in serum sodium, potassium, urea, and creatinine concentrations. For the latter, population-specific reference intervals were established and out of range values (ORV) analyzed. RESULTS: The number of AEs was similar in the spironolactone and reference groups (188 and 208, respectively), when followed for median duration of 217 days (range [2-1,333]). At each study time point, the percentage of dogs showing ORV was similar between groups. There were a higher number of deaths because of cardiac disease, renal disease or both in the reference group (30.7% versus 13.7%) (P = .0043). CONCLUSIONS AND CLINICAL IMPORTANCE: Dogs with heart failure receiving spironolactone in addition to conventional treatment are not at a higher risk for AEs, death caused by cardiac disease, renal disease, or both, hyperkalemia, or azotemia.

Effects of dietary salt intake on renal function: a 2-year study in healthy aged cats.

BACKGROUND: Increasing salt intake to promote diuresis has been suggested in the management of feline lower urinary tract disease. However, high dietary salt intake might adversely affect blood pressure and renal function. OBJECTIVES: The objective of this study was to assess the long-term effects of increased salt intake on renal function in healthy aged cats. METHODS: This study was controlled, randomized, and blinded. Twenty healthy neutered cats (10.1 ± 2.4 years) were randomly allocated into 2 matched groups. One group was fed a high salt diet (3.1 g/Mcal sodium, 5.5 g/Mcal chloride) and the other a control diet of same composition except for salt content (1.0 g/Mcal sodium, 2.2 g/Mcal chloride). Clinical examination, glomerular filtration rate, blood pressure measurement, cardiac and kidney ultrasonography, and urinary and blood tests were performed before and over 24 months after diet implementation. Statistics were performed using a general linear model. RESULTS: Sixteen cats completed the 2 year study. The only variables affected by dietary salt intake were plasma aldosterone and urinary sodium/creatinine ratio, respectively, higher and lower in the control group all over the study period and urinary specific gravity, lower in the high salt diet group at 3 months. CONCLUSIONS AND CLINICAL IMPORTANCE: Glomerular filtration rate (GFR), blood pressure, and other routine clinical pathological variables in healthy aged cats were not affected by dietary salt content. The results of this 2 year study do not support the suggestion that chronic increases in dietary salt intake are harmful to renal function in older cats.

Normal canine prostate gland: repeatability, reproducibility, observer-dependent variability of ultrasonographic measurements of the prostate in healthy intact beagles.

Most prostatic diseases in dogs are associated with prostatomegaly, and transabdominal ultrasonography has become the imaging modality of choice for evaluation of the prostate gland in the dog. The aim of the present study was to assess the reproducibility, the repeatability and interobserver variations of sonographic measurements of prostate and to determine which measurement had the lowest variability. Length and height of prostate gland were measured on longitudinal views, width of the prostate gland and height of left and right lobes of the gland on transversal views. The within-day and between-day variabilities of the prostatic parameters were determined by performing 1350 (270 length, 270 height, 270 width, 270 height of right lobe and 270 height of left lobe) examinations on ten healthy intact beagle dogs on six different days, in a two-week period (three days for the five dogs, three different days for the five others). Three observers with different levels of experience in ultrasonography performed the examinations. The lowest within-day and between-day standard deviation and coefficient of variation values were observed for the width of the prostate. The width of the gland measured on transverse frozen images seems to be the most reliable measurement for evaluating size of prostate glands in healthy dogs, although the shape, position, outline, and echogenicity of the prostate should also be assessed.

CLIP Test: a new fast, simple and powerful method to distinguish between linked or pleiotropic quantitative trait loci in linkage disequilibria analysis.

An important question arises when mapping quantitative trait loci (QTLs) for genetically correlated traits: is the correlation due to pleiotropy (a single QTL affecting more than one trait) and/or close linkage (different QTLs that are physically close to each other and influence the traits)? In this article, we propose the Close Linkage versus Pleiotropism (CLIP) test, a fast, simple and powerful method to distinguish between these two situations. The CLIP test is based on the comparison of the square of the observed correlation between a combination of apparent effects at the marker level to the minimal value it can take under the pleiotropic assumption. A simulation study was performed to estimate the power and alpha risk of the CLIP test and compare it to a test that evaluated whether the confidence intervals of the two QTLs overlapped or not (CI test). On average, the CLIP test showed a higher power (68%) to detect close-linked QTLs than the CI test (43%) and a same alpha risk (4%).

Establishing bioequivalence of veterinary premixes (Type A medicated articles).

a) Key issues concerning Premix (Type A medicated articles) Bioequivalence evaluations: 1) This is a complex issue concerning both route of administration and formulation. 2) If the animal is not at the bunk/trough, the animal is not self-administering (eating medicated feed), thus there can be no drug absorption. b) Differing opinions among scientists and regulatory authorities/expert bodies regarding: 1) No harmonization on how to design, conduct, and interpret in vivo studies. 2) Applicability of biowaivers to Type A (premix) products. 3) Why are topdress and complete feed considered differently? Are they different formulations or different routes of administration? 4) Single dose vs. multi-dose studies. 5) What is the final formulation? c) What are the next steps: 1) Harmonize current bioequivalence guidelines through the VICH process. 2) Determine the applicability/non-applicability of the Biopharmaceutical Classification System (BCS). 3) Establish the Total Mixed Ration (i.e. formulation) effects. 4) Define the test subject (individual, pen, etc.).

Renal resistive index in 55 dogs with degenerative mitral valve disease.

BACKGROUND: Azotemia occurs frequently in dogs with degenerative mitral valve disease (DMVD). It could indicate changes in renal hemodynamics. HYPOTHESIS/OBJECTIVES: To assess the renal resistive index (RI) in dogs with DMVD, and the statistical link between heart failure class, azotemia, echo-Doppler parameters, several plasma variables, and RI. ANIMALS: Fifty-five dogs with naturally occurring DVMD were used (ISACHC class 1 [n = 28], 2 [n = 19], and 3 [n = 8]). METHODS: Observational, blinded study, performed under standardized conditions. Physical examination, renal ultrasonography, and echo-Doppler examinations were performed in awake dogs. The RI of the renal, interlobar, and arcuate arteries were measured. Plasma creatinine, urea, and N-terminal pro-B-type natriuretic peptide concentrations (NT-proBNP) were determined. Statistical links between variables and RI were tested by means of a general linear model. RESULTS: Although the RI of renal and arcuate arteries were unaffected by ISACHC class, the left interlobar RI increased (P < .001) from 0.62 ± 0.05 (mean ± SD) in class 1 to 0.76 ± 0.08 in class 3. It was also higher (P < .001) in azotemic (0.74 ± 0.08) than in non-azotemic (0.62 ± 0.05) dogs. Similar findings were observed for right interlobar RI. Univariate analysis showed a positive statistical link between NT-proBNP (P = .002), urea (P < .001), creatinine (P = .002), urea-to-creatinine ratio (P < .001), left atrium-to-aorta ratio (P < .001), regurgitation fraction (P < .001), systolic pulmonary arterial pressure (P < .001), shortening fraction (P = .035), and RI. CONCLUSION AND CLINICAL IMPORTANCE: In dogs with DMVD, interlobar RI increases with heart failure severity and azotemia but a cause and effect relationship remains to be established.

Ultrasonographic evaluation of the adrenal glands in healthy dogs: repeatability, reproducibility, observer-dependent variability, and the effect of bodyweight, age and sex.

Adrenal length and width were determined from two-dimensional ultrasound longitudinal images. In study 1, 540 measurements of adrenal glands were attempted from five healthy beagle dogs by three different observers with different levels of expertise in ultrasonography, to determine the variability of adrenal gland measurements. Of these, 484 measurements were included in the statistical analysis, since 16 measurements of the left adrenal gland and 40 for the right could not be visualised by the observer. In study 2, a single measurement of both adrenal glands was taken from each of 146 dogs by the most trained observer from study 1, and the effects of different health status (healthy dogs v dogs with non-adrenal diseases), bodyweight, age and sex were assessed. A total of 267 measurements were included in the statistical analysis. The lowest intra- and inter-day coefficient of variation values were observed for the left adrenal gland and by the most trained observer. The health status had no statistically significant effect on adrenal gland length or width, whereas age had a significant effect only for the left adrenal gland (the greater the age, the greater the width or length) and sex had a significant effect only for the right adrenal gland (the width was larger in males and the length larger in females). The bodyweight had a significant effect for the length of both adrenal glands (the greater the bodyweight, the greater the length), but not the width. The differences between sd and coefficient of variation values for the width of the left adrenal gland were not statistically significant between the three observers, whereas they were statistically significant for the right adrenal gland.

Breed dependency of reference intervals for plasma biochemical values in cats.

BACKGROUND: Reference intervals (RI) are pivotal in clinical pathology. The influence of breed on RI has been poorly documented in cats. HYPOTHESIS/OBJECTIVES: RI for plasma biochemistry variables are breed-dependent in cats. ANIMALS: Five hundred and thirty-six clinically healthy, fasted, client-owned cats from 4 breeds: Holly Birman (n=132), Chartreux (n=129), Maine Coon (n=139), and Persian (n=136). METHODS: Prospective observational study: Blood samples were collected from the cephalic vein into capillary tubes containing lithium heparin. Plasma glucose, urea, creatinine, total proteins, albumin, calcium, phosphate, sodium, potassium, chloride, and total CO2 concentrations and the activities of alanine aminotransferase and alkaline phosphatase were assayed with a dry slide biochemical analyzer. RI were defined as central 95% intervals bounded by the 2.5th and 97.5th percentiles. Data were analyzed by a linear mixed effects model with type I error rate of 0.05. RESULTS: A significant (P<.05) breed effect was observed for 9/13 variables. The magnitude of the differences between breeds could be clinically relevant for creatinine, glucose, and total protein. Age, body weight, sex, and housing conditions had significant (P<.05) breed-related effects on different variables. CONCLUSIONS AND CLINICAL IMPORTANCE: Breed-specific RI should be considered for cats.

Estimation of reference intervals from small samples: an example using canine plasma creatinine.

BACKGROUND: According to international recommendations, reference intervals should be determined from at least 120 reference individuals, which often are impossible to achieve in veterinary clinical pathology, especially for wild animals. When only a small number of reference subjects is available, the possible bias cannot be known and the normality of the distribution cannot be evaluated. A comparison of reference intervals estimated by different methods could be helpful. OBJECTIVE: The purpose of this study was to compare reference limits determined from a large set of canine plasma creatinine reference values, and large subsets of this data, with estimates obtained from small samples selected randomly. METHODS: Twenty sets each of 120 and 27 samples were randomly selected from a set of 1439 plasma creatinine results obtained from healthy dogs in another study. Reference intervals for the whole sample and for the large samples were determined by a nonparametric method. The estimated reference limits for the small samples were minimum and maximum, mean +/- 2 SD of native and Box-Cox-transformed values, 2.5th and 97.5th percentiles by a robust method on native and Box-Cox-transformed values, and estimates from diagrams of cumulative distribution functions. RESULTS: The whole sample had a heavily skewed distribution, which approached Gaussian after Box-Cox transformation. The reference limits estimated from small samples were highly variable. The closest estimates to the 1439-result reference interval for 27-result subsamples were obtained by both parametric and robust methods after Box-Cox transformation but were grossly erroneous in some cases. CONCLUSION: For small samples, it is recommended that all values be reported graphically in a dot plot or histogram and that estimates of the reference limits be compared using different methods.

A new approach for the determination of reference intervals from hospital-based data.

BACKGROUND: Reference limits are some of the most widely used tools in the medical decision process. Their determination is long, difficult, and expensive, mainly because of the need to select sufficient numbers of reference individuals according to well-defined criteria. Data from hospitalized patients are, in contrast, numerous and easily available. Even if all the information required for a direct reference interval computation is usually not available, these data contain information that can be exploited to derive at least rough reference intervals. METHODS: In this article, we propose a method for the indirect estimation of reference intervals. It relies on a statistical method which has become a gold-standard in other sciences to separate components of mixtures. It relies on some distributional assumptions that can be checked graphically. For the determination of reference intervals, this new method is intended to separate the healthy and diseased distributions of the measured analyte. We assessed its performance by using simulated data drawn from known distributions and two previously published datasets (from human and veterinary clinical chemistry). RESULTS AND DISCUSSION: The comparison of results obtained by the new method with the theoretical data of the simulation and determination of the reference interval for the datasets was good, thus supporting the application of this method for a rough estimation of reference intervals when the recommended procedure cannot be used.