Curation and analysis of clinical pathology parameters and histopathologic findings from eTOXsys, a large database project (eTOX) for toxicologic studies.

Large data sharing projects amongst the pharmaceutical industry have the potential to generate new insights using data on a scale that has not been previously available. A retrospective analysis of the preclinical toxicology data collected as part of the eTOX project was conducted with the aim to provide background rates and treatment-related value analysis on both clinical pathology and histopathology datasets. Incorporated into this analysis was an extensive data consolidation task to standardise all data. Reference intervals for common clinical pathology parameters in rat and dog were generated, alongside background histopathology incidence rates in the liver, heart and kidney. Systematically applied decision thresholds allowed consistent relabelling of data points considered anomalous, and maximum fold change estimates. Relabelling of anomalous data points was conducted for the histopathology data using a Bayesian model to identify dose-dependent increases in pathologies. The results of this study allow: newly generated data to be analysed using the same methodology, rates and distributions to be used when building predictive dose-response models, and the possibility to correlate clinical pathology findings with concurrent histopathology findings. In the first half of this paper we discuss data curation, in the second half we report on the analytical methods and results.

Evaluation of novel urinary renal biomarkers: biological variation and reference change values.

A number of novel urinary biomarkers have been identified and partially qualified for use as markers for renal injury in rats. We used two multiplex assays for these novel biomarkers to quantify biomarker concentration in serial urine collections from rats of both sexes administered varying concentrations of cisplatin. From these data, we calculate inter-individual variation and reference ranges from predose animals and intra-individual variation and reference change values from undosed control animals. The biomarkers evaluated are albumin, α glutathione s-transferase, glutathione S-transferase-yb1, lipocalin-2, kidney injury molecule-1, osteopontin, and renal papillary antigen 1. For any creatinine-corrected novel biomarkers, we found intra-individual variation to be no greater than 44% and inter-individual variation to be no greater than 46%. Reference change values for most corrected analytes (except osteopontin) were 50-100%, indicating that a >100% increase in analyte concentration between serial samples would be unlikely to be associated with inherent analytical or biological variation.

Evaluation of novel urinary renal biomarkers with a cisplatin model of kidney injury: effects of collection period.

A number of novel urinary biomarkers have been identified and partially qualified for use as markers for renal injury in rats. To date, all evaluation studies have been made using 18 to 24 hour collection periods. However, shorter, more welfare friendly, urine collection periods are also used in industry. In this article, we quantify urinary biomarker concentration in serial paired sequential short and long urine collections from male rats administered varying concentrations of cisplatin. We calculate the rate of biomarker excretion in normal animals for both collection periods and the bias and correlation in urinary biomarker concentration between collection periods in dosed and control animals, and we estimate the level of agreement in biomarker concentration between both collection periods. We conclude that although there are minor differences in the concentration of some urinary biomarkers that are dependent upon the time and duration of collection, shorter collection protocols do not influence subsequent interpretation of normalized urinary biomarker data for most biomarkers.

What is your diagnosis? Preputial mass in a ferret.

: A 7-year-old neutered male polecat-type ferret (Mustela putorius furo) was presented for evaluation of a cutaneous mass close to the preputial orifice. Cytologic examination of a fine-needle aspirate revealed numerous large clumps of amorphous pink mucinous material and numerous large clumps of slightly pleomorphic epithelial cells. The cells were arranged in papillary structures, palisades, and loosely cohesive sheets with a vaguely honeycomb appearance. Occasional acinar formations were also seen. The cells had moderate to large amounts of finely granular gray to gray-blue cytoplasm. The cells were round to wispy and elongated, with indistinct borders. Often, anuclear cytoplasmic clumps were seen free in the background or adjacent to intact cells. Nuclei were round to oval and usually off-center. Chromatin was finely stippled and contained 1-3 indistinct nucleoli. Anisokaryosis and anisocytosis were moderate. Binucleated cells were noted occasionally. The cytologic features were consistent with a carcinoma of probable apocrine origin. Histopathologic examination supported a diagnosis of secretory apocrine adenocarcinoma of the preputial skin. Secretory apocrine adenocarcinomas of the prepuce are seen relatively frequently in ferrets, although their cytologic appearance has not been described widely. These neoplasms carry a poor prognosis although prompt surgical removal with wide and deep surgical margins and adjunctive radiotherapy may improve survival.

An update on FIV and FeLV test performance using a Bayesian statistical approach.

BACKGROUND: Screening tests for feline retroviruses are thought to have high sensitivity and specificity, although previous studies that evaluated these tests have limitations. Novel statistical approaches have been developed that allow the estimation of sensitivity and specificity in situations where the true state of the disease in individual animals cannot be assured. OBJECTIVE: The purpose of this study was to evaluate the sensitivity and specificity of a variety of retrovirus tests, including some screening tests, in a population of cats potentially infected with either feline leukemia virus (FeLV) and/or feline immunodeficiency virus (FIV) by using a Bayesian statistical approach. METHODS: Four hundred and ninety blood samples from cats being evaluated for FIV infection were tested by 2 rapid immunomigration tests (Witness single [WS], Witness combi [WC]) and a plate-based ELISA (Petcheck) for FIV antibody, and by a newly designed real-time polymerase chain reaction (PCR) assay for FIV provirus. Four hundred and ninety-five blood samples from cats being evaluated for FeLV infection were tested by 2 rapid immunomigration tests (WS, WC) and a plate-based ELISA (Petcheck) for FeLV antigen, and by a FeLV virus isolation technique. Results were then analyzed by using a Bayesian statistical method. RESULTS: For FIV tests, median sensitivity estimates were 0.98 for WS, 0.97 for WC, 0.98 for ELISA, and 0.92 for PCR. Median specificity estimates were 0.96 for WS, 0.96 for WC, 0.93 for ELISA, and 0.99 for PCR. For FeLV tests, median sensitivity estimates were 0.97 for WS, 0.97 for WC, 0.98 for ELISA, and 0.91 for virus isolation. Median specificity estimates were 0.96 for WS, 0.96 for WC, 0.98 for ELISA, and 0.99 for virus isolation. CONCLUSIONS: The use of Bayesian statistical methods overcomes a variety of methodologic problems associated with diagnostic test evaluations, including the lack of a definitive reference test. The sensitivity and the specificity of all 6 evaluated screening tests was high: however, specificity estimates were slightly lower than those reported by most recent studies.

Diagnosis of feline leukaemia virus infection by semi-quantitative real-time polymerase chain reaction.

In this paper the design and use of a semi-quantitative real-time polymerase chain reaction assay (RT-PCR) for feline leukaemia virus (FeLV) provirus is described. Its performance is evaluated against established methods of FeLV diagnosis, including virus isolation and enzyme-linked immunoassay (ELISA) in a population of naturally infected cats. The RT-PCR assay is found to have both a high sensitivity (0.92) and specificity (0.99) when examined by expectation maximisation methods and is also able to detect a large number of cats with low FeLV proviral loads that were negative by other conventional test methods.

Effect of chronic FIV infection, and efficacy of marbofloxacin treatment, on Mycoplasma haemofelis infection.

The purpose of this study was to investigate the effect of chronic feline immunodeficiency virus (FIV) infection, and efficacy of marbofloxacin treatment, on Mycoplasma haemofelis infection. Six cats chronically infected with FIV-Glasgow8 (Group X) and six FIV-free cats (Group Y) were infected with M. haemofelis on Day 0 by intravenous blood inoculation. From Day 0 until Day 86 post-infection (pi), blood samples were collected for M. haemofelis and FIV provirus quantitative real-time PCR and haematology. Three of the six cats in each of Groups X and Y were randomly selected to receive marbofloxacin treatment (2 mg/kg PO q24 h) from Day 16 to 43 pi, with the remaining cats being untreated controls with no antibiotic treatment. The M. haemofelis copy numbers and haematological data were compared between Groups X and Y, and between marbofloxacin-treated and control cats using a Mann-Whitney U-test. M. haemofelis infection was associated with development of macrocytic hypochromic anaemia. In some cats, marked variation in M. haemofelis copy number over time (>100,000-fold difference within 48 h in some cats) and/or cycling of copy number was seen. No correlation was found between FIV provirus copy number and M. haemofelis copy number or haematological variables. No significant effect of chronic FIV infection on M. haemofelis copy number kinetics or haematological changes due to M. haemofelis infection was found, other than MCHC (P=0.03). Marbofloxacin treatment was associated with a significant decrease in M. haemofelis copy number (P=0.002), although consistent clearance of infection was not demonstrated. This study reveals the presence of marked fluctuations in M. haemofelis copy number kinetics in vivo and a significant response to marbofloxacin antibiotic treatment.

Effect of chronic feline immunodeficiency infection, and efficacy of marbofloxacin treatment, on 'Candidatus Mycoplasma haemominutum' infection.

The purpose of this study was to investigate the effect of chronic feline immunodeficiency virus (FIV) infection, and efficacy of marbofloxacin treatment, on 'Candidatus Mycoplasma haemominutum' infection. Six cats chronically infected with FIV-Glasgow8 (group A) and six FIV-free cats (group B) were infected with 'Candidatus M. haemominutum' on day 0 by intravenous inoculation of blood. From day 0 to 105 post-infection (pi), blood samples were collected for 'Candidatus M. haemominutum' and FIV provirus quantitative real-time polymerase chain reaction (PCR) and haematological examination. Three of the six cats in each of the groups were randomly selected to receive marbofloxacin treatment (2mg/kg PO SID) from day 49 to day 76 pi, with the remaining cats being untreated controls. Maximum 'Candidatus M. haemominutum' copy number was reached around day 30 pi. No overt cycling or marked variation in copy number was observed. No significant effect of FIV infection on 'Candidatus M. haemominutum' copy number kinetics or anaemia indices was found. No correlation was found between FIV provirus copy number and 'Candidatus M. haemominutum' copy number or haematological variables. Although marbofloxacin treatment was associated with a significant decrease in 'Candidatus M. haemominutum' copy number, the copy number plateaued during treatment, with no negative PCR results. Additionally, after termination of marbofloxacin treatment the copy numbers of the treated cats increased to reach levels similar to those of the untreated cats within 7-10 days. This study documents, for the first time, the infection kinetics and antibiotic responsiveness of 'Candidatus M. haemominutum' infection.