Assessing in vitro stability of remdesivir (GS-5734) and conversion to GS-441524 in feline plasma and whole blood.

Feline infectious peritonitis (FIP) is a potentially fatal coronavirus-driven disease of cats. Treatment with nucleoside analogue GS-441524 and or prodrug remdesivir (RDV) have produced remission in both experimentally induced and naturally occurring FIP, yet information regarding metabolism of RDV into GS-441524 in cats is scarce. This study assessed possible phase I metabolism of RDV in cats, utilising an in vitro feline microsome model with in vitro t1/2 and in vitro Clint calculated using the substrate depletion method. A previously validated high-performance liquid chromatography (HPLC) fluorescence method was utilised for detection and analysis of RDV and GS-441524. Qualitative yield of RDV and intermediate metabolite GS-441524 were determined following microsome incubation, then compared to whole blood and plasma incubations. In vitro microsome incubation resulted in rapid depletion of RDV, though it did not appear to resemble a conventional phase I-dependent reaction in cats, as it is in humans and dogs. Depletion of RDV into GS-441524 was demonstrated in whole blood in vitro, suggesting cats convert RDV to GS-441524, likely via blood esterases, as observed in mice and rats. RDV metabolism is unlikely to be impacted by impaired liver function in cats. Furthermore, as RDV depletes within minutes, whereas GS-441524 is very stable, whole blood or plasma GS-441524 concentrations, rather than plasma RDV concentrations, are more appropriate for therapeutic drug monitoring (TDM) in cats receiving RDV.

Quantification of GS-441524 concentration in feline plasma using high performance liquid chromatography with fluorescence detection.

The adenosine analogue GS-441524 has demonstrated efficacy in treatment of feline infectious peritonitis (FIP). With no commercially registered formulations of GS-441524 available, global focus shifted to its pro-drug remdesivir, as it became more accessible throughout the COVID-19 pandemic. This study developed and validated a simple liquid chromatography equipped with a fluorescence detector to quantify plasma concentrations of GS-441524 applicable for routine therapeutic monitoring of remdesivir or GS-441524 therapy for FIP infected cats. A Waters X-Bridge C18, 5 µm, 150 × 4.6 mm, column was used and mixtures of 20 mM ammonium acetate (pH 4.5) with acetonitrile of 5% and 70% were prepared for gradient mobile phase. With a simple protein precipitation using methanol to clean plasma sample, GS-441524 was monitored at excitation and emission wavelengths of 250 nm and 475 nm, respectively. Using an external standard, the lowest and highest limits of quantification were 19.5 ng/mL to 10,000 ng/mL, respectively. The intra- and inter day trueness of the quality controls (QCs) were within 10% of their nominal concentrations and intra- and inter day precision of the QCs (expressed as the coefficient of variation) ranged from 1.7 to 5.7%, This assay was able to quantify plasma trough levels of GS-441524 (23.7-190.1 ng/mL) after the administration of remdesivir (9.9-15.0 mg/kg BW, IV or SC) in FIP cats (n = 12). Accordingly, this study generated an alternative and cost-effective way to quantify GS-441524 in feline biological fluids at least up to 24 hr after administrations of remdesivir.

A retrospective study on antibacterial treatments for koalas infected with Chlamydia pecorum.

Chlamydiosis remains the leading infectious disease and is one of the key factors responsible for the dramatic reduction of koala populations in South-East Queensland (SEQ) and New South Wales (NSW) regions of Australia. Possible infection outcomes include blindness, infertility, painful cystitis, and death if left untreated. Studies have reported the treatment efficacy of chloramphenicol and doxycycline, which are the two most commonly administered treatments in diseased koalas, in clinical settings. However, none have directly compared the treatment efficacy of these antibacterials on koala survival. A retrospective study was essential to identify any relationships between the demographical information, and the animals' responses to the current treatment regimens. Associations were explored between six explanatory (sex; maturity; location; clinical signs, treatment; treatment duration) and two outcome variables (survival; post-treatment PCR). Results showed that female koalas had a statistical trend of lower odds of surviving when compared to males (OR = 0.36, p = 0.05). Koalas treated with chloramphenicol for ≥ 28 days had greater odds of surviving than when treated for < 28 days (OR = 8.8, p = 0.02), and those koalas administered doxycycline had greater odds of testing PCR negative when compared to chloramphenicol treatments (OR = 5.45, p = 0.008). There was no difference between the antibacterial treatments (chloramphenicol, doxycycline, and mixed/other) and the survival of koalas. Female koalas had greater odds of exhibiting UGT signs only (OR = 4.86, p < 0.001), and also greater odds of having both ocular and UGT clinical signs (OR = 5.29, p < 0.001) when compared to males. Of the koalas, 28.5% initially had no clinical signs but were PCR positive for C. pecorum. This study enables further understanding of the complex nature between chlamydial infection and response to antibacterial treatment.

Current status on treatment options for feline infectious peritonitis and SARS-CoV-2 positive cats.

Feline infectious peritonitis (FIP) is a viral-induced, immune-mediated disease of cats caused by virulent biotypes of feline coronaviruses (FCoV), known as the feline infectious peritonitis virus (FIPV). Historically, three major pharmacological approaches have been employed to treat FIP: (1) immunomodulators to stimulate the patient's immune system non-specifically to reduce the clinical effects of the virus through a robust immune response, (2) immunosuppressive agents to dampen clinical signs temporarily, and (3) re-purposed human antiviral drugs, all of which have been unsuccessful to date in providing reliable efficacious treatment options for FIPV. Recently, antiviral studies investigating the broad-spectrum coronavirus protease inhibitor, GC376, and the adenosine nucleoside analogue GS-441524, have resulted in increased survival rates and clinical cure in many patients. However, prescriber access to these antiviral therapies is currently problematic as they have not yet obtained registration for veterinary use. Consequently, FIP remains challenging to treat. The purpose of this review is to provide an update on the current status of therapeutics for FIP. Additionally, due to interest in coronaviruses resulting from the current human pandemic, this review provides information on domesticated cats identified as SARS-CoV-2 positive.

Susceptibility of rapidly growing mycobacteria isolated from Australian cats to ivermectin, moxidectin, ceftiofur and florfenicol.

OBJECTIVES: Rapidly growing mycobacteria (RGM) infections in cats typically manifest as a panniculitis, requiring long-term antimicrobial therapy for resolution. The search for novel antimicrobial therapies to reduce treatment duration and improve the rate of clinical resolution is imperative. Accordingly, RGM isolates underwent susceptibility testing to some avermectins and other antibacterial drugs currently available. METHODS: Five Mycobacterium fortuitum and six Mycobacterium smegmatis isolates obtained from Australian cats underwent susceptibility testing by microbroth dilution to ivermectin, moxidectin, ceftiofur and florfenicol. RESULTS: All isolates were resistant to the highest concentrations of ivermectin, moxidectin and ceftiofur tested (1024 µg/ml, 256 µg/ml and 32 µg/ml, respectively). All isolates of M fortuitum were resistant to the highest concentration of florfenicol tested (128 µg/ml). The minimum inhibitory concentration range of florfenicol that inhibited growth of M smegmatis isolates was 32-64 µg/ml. CONCLUSIONS AND RELEVANCE: All drugs appear to have no efficacy in vitro for the treatment of RGM infections.

Bias in feline plasma biochemistry results between three in-house analysers and a commercial laboratory analyser: results should not be directly compared.

In-house analysers are commonplace in small animal practices but cannot be calibrated by the operator; therefore, any bias in the generated plasma analyte values cannot be corrected. Guidelines such as grading of renal disease and published reference intervals (RIs) in veterinary textbooks assume plasma biochemistry values generated by different analysers are equivalent. This study evaluated the degree of bias, as well as if bias was constant or proportional, for feline plasma biochemical analytes assessed by three in-house biochemistry analysers compared with a commercial laboratory analyser. Blood samples were collected on 101 occasions from 94 cats and, after centrifugation, plasma was divided into four aliquots. One aliquot was sent to the commercial laboratory and the remaining three were tested using the in-house biochemistry analysers. Results from each analyser were compared with the commercial laboratory results by difference plots and analyses, and by comparing percentages of results within provided RIs. Substantial bias was evident relative to the results of the commercial analyser for at least half of the analytes tested for each machine. In most cases, bias was proportional, meaning that the difference between the methods varied with the concentration of the analyte. The results demonstrate that values obtained from these analysers should not be directly compared and that RIs are not transferable between these analysers. Potential effects of bias on clinical decision-making may be overcome by use of appropriately generated RIs, or reference change values which, for most biochemistry analytes, are more appropriate than subject-based RIs.

Assessments of feline plasma biochemistry reference intervals for three in-house analysers and a commercial laboratory analyser.

For each species, the manufacturers of in-house analysers (and commercial laboratories) provide standard reference intervals (RIs) that do not account for any differences such as geographical population differences and do not overtly state the potential for variation between results obtained from serum or plasma. Additionally, biases have been demonstrated for in-house analysers which result in different RIs for each different type of analyser. The objective of this study was to calculate RIs (with 90% confidence intervals [CIs]) for 13 biochemistry analytes when tested on three commonly used in-house veterinary analysers, as well as a commercial laboratory analyser. The calculated RIs were then compared with those provided by the in-house analyser manufacturers and the commercial laboratory. Plasma samples were collected from 53 clinically normal cats. After centrifugation, plasma was divided into four aliquots; one aliquot was sent to the commercial laboratory and the remaining three were tested using the in-house biochemistry analysers. The distribution of results was used to choose the appropriate statistical technique for each analyte from each analyser to calculate RIs. Provided reference limits were deemed appropriate if they fell within the 90% CIs of the calculated reference limits. Transference validation was performed on provided and calculated RIs. Twenty-nine of a possible 102 provided reference limits (28%) were within the calculated 90% CIs. To ensure proper interpretation of laboratory results, practitioners should determine RIs for their practice populations and/or use reference change values when assessing their patients' clinical chemistry results.

Biological variation and reference change values of feline plasma biochemistry analytes.

This is the first report concerning biological variation and reference change values of feline plasma biochemistry components in the peer-reviewed literature. Biological variation refers to inherent physiological variation of analytes. The ratio of individual biological variation to group biological variation is referred to as an analyte's index of individuality. This index determines the suitability of an analyte to be assessed in relation to population- or subject-based reference intervals. A subject-based reference interval is referred to as a reference change value or critical difference, and is calculated from individual biological variation. Fourteen cats were sampled for plasma biochemistry analysis once weekly for 6 weeks. Samples were stored and then tested at the same time. Results were assessed in duplicate and coefficients of variation for each analyte were isolated to distinguish variation within each subject, between all subjects and by the analyser. From these results, an index of individuality and reference change values were determined for each analyte. Five plasma biochemistry analytes (alkaline phosphatase, alanine aminotransferase, cholesterol, creatinine and globulin) had high individuality and, therefore, subject-based reference intervals are more appropriate; only one analyte (sodium) had low individuality, indicating that population-based reference intervals are appropriate. Most analytes had intermediate individuality so population-based reference intervals should be assessed in relation to subject-based reference intervals. The results of this study demonstrate high individuality for most analytes and, therefore, that population-based reference intervals are of limited utility for most biochemical analytes in cats.

Preliminary post-prandial studies of Burmese cats with elevated triglyceride concentrations and/or presumed lipid aqueous.

A proportion of Burmese cats in Australia have an exaggerated post-prandial triglyceride (TG) response after an oral fat tolerance test (OFTT). The aim of this study was to determine (a) whether Burmese cats with presumed lipid aqueous (PLA) had exaggerated post-prandial triglyceridaemia, (b) if Burmese cats with exaggerated post-prandial triglyceridaemia ('affected' cats) had decreased lipoprotein lipase (LPL) activity and (c) whether affected cats were more insulin resistant than normal Burmese cats. Of cats with a history of PLA, 4/5 were shown to be lipid intolerant (4h TG>4.5mmol/l). Four affected Burmese cats were age, gender and body condition matched to four normal Burmese cats. Serum TG, insulin, non-esterified fatty acids (NEFA), lipoprotein and apolipoprotein concentrations were determined 2 weeks after commencing a standardised high-protein diet, with an OFTT performed 4 weeks later. Affected Burmese cats did not have significantly different fasting insulin, fructosamine, NEFA, apolipoprotein or lipoprotein concentrations compared to control cats. During the OFTT, affected cats had significantly higher 4h and 6h serum TG and NEFA concentrations than normal cats. There was a trend for lower LPL activity, higher insulin concentrations (at 4 and 6h) and higher insulin area under the curve (AUC) during the OFTT in affected Burmese cats compared to controls, although these results failed to reach significance, probably due to the small number of cats studied. Further investigations using larger numbers of cats should focus on reduced LPL activity and insulin resistance as potential causes of delayed TG clearance.

Assessment of the Accutrend GCT and PTS CardioChek meters to measure blood triglyceride concentrations in cats.

Point-of-care (POC) meters that determine whole blood triglyceride (TG) concentrations are used in human medicine to monitor both fasting and post-prandial TG concentrations. The aim of this study was to evaluate their performance for determining feline TG concentrations. A total of 116 venous blood samples were collected from 55 cats. TG concentrations were measured in whole blood using two meters: the Accutrend glucose cholesterol triglyceride (GCT) (GCT: Roche Diagnostics) and PTS CardioChek (PTS - Polymer Technology Systems), and results compared to those determined by a National Association of Testing Authorities (NATA) accredited veterinary laboratory. The GCT was not suitable for use in cats with normal TG concentrations (<0.9 mmol/l), as it overestimated almost 80% of the values; however, this device performed better with TG concentrations between 0.9 and 2.0 mmol/l. The PTS meter performed well in cats with normal TG concentrations, correctly classifying 90% of values as 'normal', and fairly well with TG concentrations <2.0 mmol/l. The PTS meter could be used to determine whether cats have normal fasting TG concentrations or predict mild elevations in serum TG, whereas the GCT meter can only be used to predict cats with elevated TG concentrations. Although both meters have limitations in determining some TG concentrations, the PTS in particular, could be used as a screening tool to distinguish normal cats to those with hypertriglyceridaemia.

Permethrin spot-on intoxication of cats Literature review and survey of veterinary practitioners in Australia.

UNLABELLED: SURVEY AIMS: A questionnaire was sent to veterinarians in Australia to determine the approximate number of cats presenting for permethrin spot-on (PSO) intoxication over a 2-year period. FINDINGS: Of the 269 questionnaires returned, 255 were eligible for analysis. A total of 207 respondents (81%) reported cases of PSO intoxication in cats over the previous 2 years. In total, 750 individual cases were reported, with 166 deaths. While all deaths were generally attributable to intoxication, 39 cats were euthanased because owners were unable to pay the anticipated treatment costs. Brands of PSO implicated included Exelpet Flea (and Tick) Liquidator (Mars Australia) (146 respondents), Bayer Advantix (48), Purina Totalcare Flea Eliminator Line-On (19), Troy Ease-On (six) and Duogard Line-On (Virbac) (four); 67 respondents were not able to identify a specific product. Permethrin spot-on formulations were most commonly obtained from supermarkets (146 respondents), followed by pet stores (43), veterinary practices (16), and a range of other sources including produce stores and friends. The majority of intoxication cases reported involved PSOs labelled for use in dogs with specific label instructions such as 'toxic to cats'. Owners applied these PSO products to their cats accidentally or intentionally. In some cases, exposure was through secondary contact, such as when a PSO product was applied to a dog with which a cat had direct or indirect contact. RECOMMENDATIONS: In the authors' view, because of the likelihood of inappropriate use and toxicity in the non-labelled species, over-the-counter products intended for use in either dogs or cats must have a high margin of safety in all species. Furthermore, PSOs should only be available at points of sale where veterinary advice can be provided and appropriate warnings given. As an interim measure, modified labelling with more explicit warnings may reduce morbidity and mortality.

Triglyceride response following an oral fat tolerance test in Burmese cats, other pedigree cats and domestic crossbred cats.

Primary lipid disorders causing fasting triglyceridaemia have been documented infrequently in Burmese cats. Due to the known increased risk of diabetes mellitus and sporadic reports of lipid aqueous in this breed, the aim of this study was to determine whether healthy Burmese cats displayed a more pronounced pre- or post-prandial triglyceridaemia compared to other cats. Serum triglyceride (TG) concentrations were determined at baseline and variably at 2, 4 and 6h after ingestion of a high-fat meal (ie, an oral fat tolerance test) in a representative sample of Burmese and non-Burmese cats. The median 4 and 6h serum TG concentrations were significantly higher in Burmese cats (4h - 2.8mmol/l; 6h - 8.2mmol/l) than in other pedigree and domestic crossbred cats (4h - 1.5mmol/l; 6h - 1.0mmol/l). The non-Burmese group had post-prandial TG concentrations ranging from 0.6 to 3.9mmol/l. Seven Burmese cats had post-prandial TG concentrations between 6.6 and 19.0mmol/l, five had concentrations between 4.2 and 4.7mmol/l, while the remaining 15 had post-prandial concentrations between 0.5 and 2.8mmol/l. None of these Burmese cats had fasting triglyceridaemia. Most Burmese cats with a 4 h TG > 6.0 mmol/l had elevated fasting very low density lipoprotein (VLDL) concentrations. This study demonstrates that a proportion of Burmese cats in Australia have delayed TG clearance compared to other cats. The potential repercussions of this observation with reference to lipid aqueous, pancreatitis and diabetes mellitus in Burmese cats are discussed.