Laboratory evaluation of a rapid diagnostic test for dairy mastitis.

Rapid diagnostic tests that differentiate between Gram positive, Gram negative and the absence of aerobic bacteria in milk samples from dairy cows with clinical mastitis can support antimicrobial treatment decisions and contribute to a more prudent use of antimicrobials in the dairy industry. The objective of this study was to evaluate the test characteristics of the novel rapid BACT mastitis test in discriminating causes of clinical mastitis under laboratory conditions. Test outcomes of 155 milk samples from clinical mastitis cases were incubated for 14-16 h in the BACT test and compared to results of bacteriological culture. The accuracy for detection of bacterial growth and Gram positive growth was 91 and 89%, respectively. The BACT test could provide an accurate and relatively fast decision tool for farmers to aid in antimicrobial treatment decisions in cases of clinical mastitis.

Evaluation of Bayesian Hui-Walter and logistic regression latent class models to estimate diagnostic test characteristics with simulated data.

Estimation of the accuracy of diagnostic tests in the absence of a gold standard is an important research subject in epidemiology (Dohoo et al., 2009). One of the most used methods the last few decades is the Bayesian Hui-Walter (HW) latent class model (Hui and Walter, 1980). However, the classic HW models aggregate the observed individual test results to the population level, and as a result, potentially valuable information from the lower level(s) is not fully incorporated. An alternative approach is the Bayesian logistic regression (LR) latent class model that allows inclusion of individual level covariates (McInturff et al., 2004). In this study, we explored both classic HW and individual level LR latent class models using Bayesian methodology within a simulation context where true disease status and true test properties were predefined. Population prevalences and test characteristics that were realistic for paratuberculosis in cattle (Toft et al., 2005) were used for the simulation. Individual animals were generated to be clustered within herds in two regions. Two tests with binary outcomes were simulated with constant test characteristics across the two regions. On top of the prevalence properties and test characteristics, one animal level binary risk factor was added to the data. The main objective was to compare the performance of Bayesian HW and LR approaches in estimating test sensitivity and specificity in simulated datasets with different population characteristics. Results from various settings showed that LR models provided posterior estimates that were closer to the true values. The LR models that incorporated herd level clustering effects provided the most accurate estimates, in terms of being closest to the true values and having smaller estimation intervals. This work illustrates that individual level LR models are in many situations preferable over classic HW models for estimation of test characteristics in the absence of a gold standard.

Neurologic Assessment and Critical Care of Exotic Animals: Approach to the Neurologic Exam, Species Differences, Prognostic Scales, Commonly Encountered Conditions, Ancillary Diagnostic Tests, and Caring for Neurologically Impaired Patients.

Many disorders of other body systems have been well characterized in exotic species; however, data regarding neurologic conditions is limited. Across some of these species, correlates between feline and canine neurology can be made, but variations in the nervous system anatomy make evaluation more challenging. With accurate neurolocalization a focused list of differential diagnoses can be created. Performing the neurologic examination should be methodical for all patients, and the order and extent of examination may depend upon the patient's clinical condition and cooperation. Applications of objective scale measures (such as coma scales), and ancillary diagnostics (electrodiagnostics, advanced imaging, biopsy techniques, and BAER testing) complement physical assessment and clinicopathologic assessment in these neurologic patients. Once a neurolocalization, likely diagnosis, and prognosis have been established, specific considerations for hospitalization and care of neurologic patients can be implemented while treatment is instituted.

Profile likelihood confidence interval for the prevalence assessed by an imperfect diagnostic test.

We present a new confidence interval for the prevalence of a disease for a situation when sensitivity and specificity of the diagnostic test are estimated from validation samples independent of the study sample. The new interval is based on profile likelihood and incorporates an adjustment improving the coverage probability. Its coverage probability and expected length were assessed by simulation and compared to two other methods for this problem, namely those by Lang and Reiczigel (2014) and Flor et al. (2020). Expected length of the new interval is less than that of the Lang and Reiczigel interval while its coverage is about the same. Comparison to the Flor interval resulted in similar expected length but higher coverage probabilities for the new interval. All in all, the new interval proved to be better than both its competitors.

Interpretation and Analysis of Individual Diagnostic Tests and Performance.

Diagnostic tests are performed daily by bovine practitioners at the individual and population level. At the individual level, they help not only for making a diagnosis, but can also serve to rule in or rule out a specific condition, monitor treatment response, establish a prognosis, or to determine infection status. Performing an individual diagnostic test is technical; however, its interpretation and contextualization requires medical and epidemiologic skills that veterinary practitioners are able to master. This article shows the added value of the context of test prescription and correct interpretation highlighting the central role of the veterinary practitioner.

Ultrasonographic monitoring of feline epaxial muscle height as part of an annual wellness examination to assess for the development of sarcopenia.

OBJECTIVES: The aim of this study was to determine if epaxial muscle height (EMH) could be reliably incorporated into annual routine wellness screenings, and also determine its relationship to age, body condition score (BCS), subjective muscle assessment (SMA), breed and sex in mature cats. METHODS: EMH was determined independently by three observers from ultrasonographic examinations - collected by an additional trained individual - of cats enrolled at the Feline Healthy Ageing Clinic, University of Liverpool, UK. Age, body weight, BCS and SMA data were also collected. RESULTS: A total of 92 cats were included, 35 of which had repeat ultrasonographic examinations 12 months apart. Enrolled cats were a median age of 8 years and 9 months at the time of the first measurement. Variation in the quality of ultrasonographic images collected did not affect muscle depth measurements (P = 0.974). Further, there was good intra- and inter-observer repeatability for all observations (intraclass correlation range 0.97-0.99). There was a moderate positive association between EMH and body weight (r = 0.49, P <0.001) but no association with age (r = -0.05, P = 0.680). There were also positive associations in EMH among cats with different BCSs (P = 0.001) and SMAs (thoracic spine, P = 0.021; lumbar spine, P = 0.014), but breed (P = 0.429) and sex (P = 0.187) had no effect. Finally, there was no change in EMH measurements in the paired samples (P = 0.145) or correlation between percentage weight and EMH change over 12 months. CONCLUSIONS AND RELEVANCE: The accuracy of EMH measurement using ultrasonographic imaging is good, irrespective of observer experience and provided that the ultrasonographer has some training. This suggests that ultrasonographic measurement of EMH could have a major practical impact as a non-invasive determination of muscle mass in pet cat populations. Further research is required to assess longitudinal changes in muscle mass over time in senior pet cats.

Characteristics (sensitivity and specificity) of herd-level diagnostic tests for Mycobacterium avium subspecies paratuberculosis in cattle - A systematic review.

Diagnostic tests for Johne's disease in cattle are characterised by poor sensitivity and often imperfect specificity at the animal level. Because farmers and veterinarians have limited assurance or confidence from results of testing individual animals for Mycobacterium avium subsp. paratuberculosis (MAP), control programmes based on whole herd-level testing provide the best framework for classifying herds. At the herd level, there is a diverse range of testing options for MAP based on both direct and indirect testing of individual and pooled samples. The most common measures of herd test performance, herd sensitivity (HSe) and herd specificity (HSp), are important for decision-making in herd test selection, for estimating prevalence and as inputs for simulation studies. This systematic review investigated the results of herd test evaluations for MAP in cattle, through a comprehensive search of the literature and a systematic four-stage screening process to identify relevant publications. Forty-six publications with relevant results were eligible for inclusion in the final review, containing evaluations of whole-herd ELISA serological testing, bulk milk tank ELISA, culture, PCR and phage testing, pooled faecal testing and environmental sample testing. Data extracted from each publication included sample populations, methods of analysis, reference tests, cut-off points, HSe and HSp. Direct comparisons between the reported HSe and HSp estimates of different studies is challenging due to the variations in herd prevalence and test protocols used. The data in this systematic review will benefit decision-makers and researchers and highlights knowledge gaps requiring further research.

Lateral flow devices for samples collected by straw sampling method for postmortem canine rabies diagnosis.

The direct fluorescent antibody test (dFAT) using brain sample after opening the skull is the standard rabies diagnostic test in animal rabies. However, it is not feasible in many resource-limited settings. Lateral flow devices (LFD) combined with a simple sampling methodology is quicker, simpler, and less hazardous than the standard test and can be a useful tool. We conducted a prospective on-site study to evaluate the diagnostic accuracy of the LFD with the straw sampling method compared with that of the dFAT with the skull opening procedure for post-mortem canine rabies diagnosis. We collected 97 rabies-suspected animals between December 1, 2020 and March 31, 2021. Among the 97 samples, 53 and 50 cases were positive tests for dFAT and LFD, respectively. The sensitivity and specificity of LFD with straw sampling method were 94.3% (95% confidence interval [CI], 84.3-98.8%) and 100% (95% CI, 92.0-100%), respectively. The performance of LFD by the straw sampling method showed relatively high sensitivity and 100% specificity compared with that of dFAT performed on samples collected after opening the skull. This methodology can be beneficial and is a strong tool to overcome limited animal surveillance in remote areas. However, because of our limited sample size, more data using fresh samples on-site and the optimizations are urgently needed for the further implementation in endemic areas.

On-farm colorimetric detection of Pasteurella multocida, Mannheimia haemolytica, and Histophilus somni in crude bovine nasal samples.

This work modifies a loop-mediated isothermal amplification (LAMP) assay to detect the bovine respiratory disease (BRD) bacterial pathogens Pasteurella multocida, Mannheimia haemolytica, and Histophilus somni in a colorimetric format on a farm. BRD causes a significant health and economic burden worldwide that partially stems from the challenges involved in determining the pathogens causing the disease. Methods such as polymerase chain reaction (PCR) have the potential to identify the causative pathogens but require lab equipment and extensive sample processing making the process lengthy and expensive. To combat this limitation, LAMP allows accurate pathogen detection in unprocessed samples by the naked eye allowing for potentially faster and more precise diagnostics on the farm. The assay developed here offers 66.7-100% analytical sensitivity, and 100% analytical specificity (using contrived samples) while providing 60-100% concordance with PCR results when tested on five steers in a feedlot. The use of a consumer-grade water bath enabled on-farm execution by collecting a nasal swab from cattle and provided a colorimetric result within 60 min. Such an assay holds the potential to provide rapid pen-side diagnostics to cattle producers and veterinarians.

Bayesian latent class analysis when the reference test is imperfect.

Latent class analysis (LCA) has allowed epidemiologists to overcome the practical constraints faced by traditional diagnostic test evaluation methods, which require both a gold standard diagnostic test and ample numbers of appropriate reference samples. Over the past four decades, LCA methods have expanded to allow epidemiologists to evaluate diagnostic tests and estimate true prevalence using imperfect tests over a variety of complex data structures and scenarios, including during the emergence of novel infectious diseases. The objective of this review is to provide an overview of recent developments in LCA methods, as well as a practical guide to applying Bayesian LCA (BLCA) to the evaluation of diagnostic tests. Before conducting a BLCA, the suitability of BLCA for the pathogen of interest, the availability of appropriate samples, the number of diagnostic tests, and the structure of the data should be carefully considered. While formulating the model, the model's structure and specification of informative priors will affect the likelihood that useful inferences can be drawn. With the growing need for advanced analytical methods to evaluate diagnostic tests for newly emerging diseases, LCA is a promising field of research for both the veterinary and medical disciplines.

L'analyse à classes latentes a permis aux épidémiologistes de surmonter les problèmes concrets posés par les méthodes traditionnelles d'évaluation des essais de diagnostic, qui nécessitent à la fois un test de référence absolue (étalon ou gold standard) et un grand nombre d'échantillons de référence aux caractéristiques appropriées. Au cours des quatre dernières décennies, les méthodes d'analyse à classes latentes ont acquis de l'ampleur et permettent aux épidémiologistes d'évaluer les essais diagnostiques et d'estimer les taux de prévalence réelle tout en recourant à des tests supposés imparfaits, grâce à l'utilisation de données et de scénarios divers et complexes, y compris dans les situations d'émergence de nouvelles maladies infectieuses. Les auteurs font un tour d'horizon des dernières évolutions dans ce domaine et donnent des orientations pratiques concernant la manière d'utiliser l'analyse bayésienne à classes latentes pour évaluer les performances d'un test de diagnostic. Avant de conduire une telle analyse, il convient de déterminer avec soin si elle est adaptée à l'agent pathogène considéré et si les échantillons disponibles sont appropriés et en nombre suffisant ; il convient également de prendre en compte le nombre de tests de diagnostic à évaluer et la structure des données utilisées. Lors de la conception du modèle, sa structure et la définition préalable des données informatives vont affecter la probabilité que le modèle génère des inférences utiles. Face à la nécessité croissante de disposer de méthodes analytiques sophistiquées pour évaluer les tests de diagnostic utilisés pour les maladies émergentes nouvelles, les analyses à classes latentes offrent des perspectives prometteuses pour la recherche, aussi bien dans le domaine de la santé vétérinaire que de la médecine humaine.

El análisis de clases latentes ha servido a los epidemiólogos para superar las limitaciones prácticas que imponen los métodos tradicionales de evaluación de pruebas de diagnóstico, que requieren a la vez una prueba de diagnóstico que sirva de patrón de referencia perfecto y un gran número de muestras de referencia adecuadas. En los últimos cuatro decenios, los métodos de análisis de clases latentes se han ido ampliando hasta permitir a los epidemiólogos evaluar pruebas de diagnóstico y calcular la prevalencia real empleando pruebas imperfectas ante muy diversas estructuras de datos y situaciones complejas, incluida la aparición de nuevas enfermedades infecciosas. Los autores, tras presentar a grandes líneas los últimos adelantos en cuanto a métodos de análisis de clases latentes, ofrecen indicaciones prácticas para aplicar el análisis bayesiano de clases latentes a la evaluación de pruebas de diagnóstico. Antes de proceder a un análisis bayesiano de este tipo conviene estudiar con detenimiento la idoneidad del método para el patógeno en cuestión, la disponibilidad de muestras apropiadas, el número de pruebas de diagnóstico y la estructura de los datos. A la hora de formular el modelo, la estructura del propio modelo y la especificación de los elementos informativos previos influirán en la probabilidad de poder extraer conclusiones provechosas. Ante la creciente necesidad de disponer de métodos analíticos avanzados con los que evaluar pruebas de diagnóstico de nuevas enfermedades emergentes, el análisis de clases latentes abre un promisorio campo de investigación para las disciplinas veterinarias y médicas.

Diagnostic test validation studies when there is a perfect reference standard.

Before tools became available to consider diagnostic test validation studies where a 'gold-standard' is not available, new diagnostic tests were compared to a reference standard assumed to be highly accurate if not perfect. This paper reviews such 'traditional' situations with examples and methods of study design and analysis. Three situations are described, two where a perfect reference is available for either positive or negative animals, and one where the reference is perfect for both. Thus, here the authors review circumstances to be considered when validating a diagnostic test with a credible reference standard. An appropriate study design requires an unbiased selection of animals from the population to which a new test will be applied. Examples for calculating sample size and data analysis are provided. Finally, the authors discuss situations where it may be appropriate to include influential variables ('covariates') in a diagnostic test validation study..

Avant la mise au point d'outils permettant de concevoir des études de validation pour des tests candidats en l'absence d'un étalon de référence à l'exactitude parfaite (référence absolue ou gold standard), les tests à valider étaient comparés à un étalon de référence censé présenter un niveau élevé d'exactitude, à défaut d'être parfait. Les auteurs décrivent ces situations « classiques ¼ en donnant quelques exemples et en précisant les méthodes employées pour la conception et l'analyse de ces études. Ils décrivent trois situations : dans deux d'entre elles, un étalon de référence parfait existe, soit pour les animaux positifs, soit pour les animaux négatifs ; dans la troisième situation, l'étalon de référence est parfait pour les deux catégories. Ainsi, les circonstances prises en compte ici sont celles de la validation d'un test diagnostique au moyen d'un réactif de référence crédible. Une conception d'étude appropriée passe par une sélection non biaisée des individus composant l'échantillon au sein de la population animale à laquelle sera appliqué le test. Les auteurs donnent quelques exemples de calcul de la taille de l'échantillon et d'analyse des données. Enfin, ils examinent les situations où il peut être opportun d'inclure des variables influentes (« covariance ¼) dans l'étude de validation d'un test diagnostique.

Antes de que apareciesen herramientas que permiten juzgar los estudios de validación de pruebas de diagnóstico en ausencia de un modelo o patrón de calibración, las nuevas pruebas de diagnóstico eran comparadas con un patrón de referencia considerado muy exacto, cuando no perfecto. Los autores se refieren aquí a este tipo de situaciones "clásicas", exponiendo ejemplos y métodos de diseño de estudios y análisis de los datos obtenidos. Los autores describen tres tipos de situación: cuando se dispone de una referencia perfecta para animales positivos; cuando se dispone de una referencia perfecta para animales negativos; y cuando la referencia es perfecta para ambos casos. Para cada tipo de situación examinan los aspectos que hay que tener en cuenta al validar una prueba de diagnóstico con un patrón de referencia fiable. El diseño adecuado de un estudio exige una selección no sesgada de los animales a partir de una población a la que vaya a aplicarse la nueva prueba. Los autores ofrecen ejemplos del modo de calcular el tamaño muestral y analizar los datos de un estudio. Por último, examinan situaciones en las que pueda ser conveniente incluir en el estudio de validación uno o más factores que puedan influir (covariables).

Examples of proper reporting for evaluation (Stage 2 validation) of diagnostic tests for diseases listed by the World Organisation for Animal Health.

Reporting and design standards are key indicators of the quality of diagnostic accuracy (validation) studies but, with the exception of aquatic animal diseases and paratuberculosis in ruminants, there is limited guidance for designing these studies in animals. There is, therefore, a need for generic guidelines that are based on disease characteristics, such as mode of transmission, latent period and pathogenesis. Comprehensive, clear and transparent reporting of primary test accuracy studies for diseases listed by the World Organisation for Animal Health (OIE) has value for the end users of diagnostic tests and, ultimately, for decision-makers, who require systematic reviews and meta-analysis of multiple tests for specified diseases and testing purposes. The recent publication of reporting standards for Bayesian latent class models, to analyse test-accuracy data from naturally occurring disease events, fills an important gap as these methods are being increasingly used for OIE-listed diseases. Adherence to design and reporting standards, as well as to guidelines, helps to ensure that research funding for test validation studies is used appropriately and that the strengths and limitations of single tests or test combinations are made clear to test users. The authors provide a review of key points that are often overlooked or misinterpreted in test validation studies, as well as two concrete examples of good practice for use as a reference point for future studies.

Les normes de notification et de conception sont des indicateurs essentiels de la qualité des études de validation des tests destinées à déterminer leur exactitude diagnostique ; or, en dehors des maladies des animaux aquatiques et de la paratuberculose chez les ruminants, il n'existe guère de lignes directrices pour concevoir ce type d'études pour les tests utilisés en santé animale. À la connaissance des auteurs, il n'existe pas non plus de normes de conception applicables aux études de validation en santé humaine. Par conséquent, il conviendrait de disposer de lignes directrices génériques fondées sur les caractéristiques des maladies telles que leurs modalités de transmission, leur période de latence et leur pathogénie. Une notification complète, claire et transparente des études d'exactitude des tests primaires pour les maladies listées par l'Organisation mondiale de la santé animale (OIE) serait une aide précieuse pour les utilisateurs finaux des tests de diagnostic, mais aussi pour les responsables de l'élaboration des politiques, dont les décisions reposent sur des examens et des méta-analyses systématiques couvrant un grand nombre de tests pour certaines maladies ou pour certains usages d'un test. La publication récente des normes de notification applicables aux modèles bayésiens à classe latente pour analyser les données de performance d'un test à partir de foyers naturels de maladie comble une lacune importante dans la mesure où ces méthodes sont de plus en plus utilisées pour les maladies listées par l'OIE. L'adhésion à des normes de conception et de notification ainsi qu'à des lignes directrices en la matière permettra de garantir que les fonds alloués aux études de validation des tests sont bien utilisés et que les atouts et les limitations de certains tests individuels ou associations de tests sont clairement perçus par les utilisateurs. Les auteurs passent en revue certains points essentiels qui sont souvent ignorés ou mal interprétés lors des études de validation des tests et proposent deux exemples concrets de bonnes pratiques qui pourront servir de références pour les études à venir.

Las normas de comunicación y diseño son indicadores básicos de la calidad de los estudios encaminados a determinar la exactitud de diagnóstico (validación) pero, con la excepción de las enfermedades de los animales acuáticos y la paratuberculosis en rumiantes, hay escasas directrices que se apliquen al diseño de esos estudios en animales. Además, hasta donde saben los autores, en el ámbito de la salud humana no hay normas de diseño. De ahí la necesidad de directrices genéricas que estén basadas en las características de las enfermedades, como modo de transmisión, período de latencia o patogénesis. La comunicación exhaustiva, clara y transparente de estudios primarios sobre la exactitud de pruebas de diagnóstico de enfermedades incluidas en las listas de la Organización Mundial de Sanidad Animal (OIE) reviste utilidad no solo para los usuarios finales de la prueba, sino también, en última instancia, para los órganos decisorios, que necesitan metaanálisis y estudios sistemáticos de múltiples pruebas que se apliquen a una u otra enfermedad y sirvan para una u otra finalidad. La reciente publicación de normas de comunicación de modelos bayesianos de clases latentes para analizar los datos de exactitud de pruebas a partir de episodios de enfermedad de origen natural viene a colmar una importante laguna, en la medida en que estos métodos se aplican cada vez más al diagnóstico de enfermedades incluidas en las listas de la OIE. El cumplimiento de las normas de diseño y comunicación, y también de las directrices, ayuda a garantizar que los fondos de investigación destinados a estudios de validación de pruebas sean utilizados debidamente y que el usuario final de una prueba reciba información clara sobre los puntos fuertes y las limitaciones de una prueba o combinación de pruebas. Los autores pasan revista a los principales aspectos que se suelen pasar por alto o malinterpretar en los estudios de validación de pruebas y ofrecen dos ejemplos concretos de buenas prácticas que se pueden utilizar como referencia en futuros estudios.

Comparison of diagnostic methods for Tetracapsuloides bryosalmonae detection in salmonid fish.

Diagnostic accuracy of pathogen detection depends upon the selection of suitable tests. Problems can arise when the selected diagnostic test gives false-positive or false-negative results, which can affect control measures, with consequences for the population health. The aim of this study was to compare sensitivity of different diagnostic methods IHC, PCR and qPCR detecting Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease in salmonid fish and as a consequence differences in disease prevalence. We analysed tissue from 388 salmonid specimens sampled from a recirculating system and rivers in the Czech Republic. Overall prevalence of T. bryosalmonae was extremely high at 92.0%, based on positive results of at least one of the above-mentioned screening methods. IHC resulted in a much lower detection rate (30.2%) than both PCR methods (qPCR32: 65.4%, PCR: 81.9%). While qPCR32 produced a good match with IHC (60.8%), all other methods differed significantly (p < .001) in the proportion of samples determined positive. Both PCR methods showed similar sensitivity, though specificity (i.e., the proportion of non-diseased fish classified correctly) differed significantly (p < .05). Sample preservation method significantly (p < .05) influenced the results of PCR, with a much lower DNA yield extracted from paraffin-embedded samples. Use of different methods that differ in diagnostic sensitivity and specificity resulted in random and systematic diagnosis errors, illustrating the importance of interpreting the results of each method carefully.

Measures of diagnostic precision (repeatability and reproducibility) for three test methods designed to detect spring viremia of carp virus.

Spring viremia of carp virus (SVCV) is a rhabdovirus of the Sprivivirus genus and the etiological agent of an internationally regulated aquatic animal disease in several fish species, including koi carp Cyprinus carpio L. The virus has a complex lifecycle with both acute and persistent stages of infection and can cause high mortality in affected populations. In this study, the diagnostic repeatability (within laboratory agreement) and reproducibility (between laboratory agreement) of 3 tests were investigated to assess their fitness as SVCV diagnostic tools. The tests, reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays targeting either the SVCV glycoprotein (Q1G) or nucleoprotein (Q2N) genes and virus isolation by cell culture (VI), were performed in a blinded study with four Canadian laboratories. Test panels consisted of duplicate sets of 100 tissue samples collected from 3 SVCV prevalence populations of koi: a low-prevalence negative reference population (n = 20 fish) as well as moderate- (n = 50 fish) and high-prevalence (n = 30 fish) populations of koi experimentally infected with SVCV. The Q1G and Q2N tests were performed with kidney tissue in 3 laboratories and with brain tissue in 1 laboratory whereas pools of kidney, spleen and gill tissues were tested with the VI assay in 2 laboratories. Agreement of binary results was evaluated using the observed proportion of agreement, Cohen's kappa and Gwet's agreement coefficient (AC1) whereas the concordance correlation coefficient (ccc) and Bland Altman's limit of agreement were used to evaluate agreement of the RT-qPCR continuous data. Gwet's AC1 provided a more stable estimate of agreement than Cohen's kappa. Overall, high repeatability (AC1, 0.78-0.90) and reproducibility (AC1, 0.74-0.89) were observed for the Q1G and Q2N tests when kidney tissue was used. Lower agreement estimates of repeatability (AC1, 0.54-0.77) and reproducibility (AC1, 0.50-0.80) were obtained for the VI test. RT-qPCR reproducibility was low with kidney-brain tissue pairs (AC1, 0.09-0.46) and high with inter-test pairs of brain (AC1, 0.76-0.86) or kidney tissue (0.75-0.86). Tissue-specific differences in virus load affected test precision and informed final tissue selection. Repeatability (ccc, 0.94-0.97) and reproducibility (ccc, 0.91-0.97) estimates of agreement for paired continuous data from the RT-qPCR assays were similarly high with kidney tissue and lower with paired brain (ccc, 0.15-0.83) and kidney-brain tissues (ccc, 0.01-0.55). The high precision of Q1G and Q2N with kidney tissue suggests that the tests are performing similarly and are suitable candidates for assessment of their diagnostic accuracy.

Accurate and rapid detection of Fasciola hepatica copro-DNA in sheep using loop-mediated isothermal amplification (LAMP) technique.

Fascioliasis is a parasitic infection caused by Fasciola spp. in humans and animals. Despite significant advances in vaccination and new therapeutic agents, little attention has been paid to validate methods for the diagnosis of fascioliasis in animals. This study aimed to compare the loop-mediated isothermal amplification (LAMP) technique with PCR assay for the diagnosis of F. hepatica in sheep. In this cross-sectional study, 195 stool samples were collected from sheep for 3 months in Lorestan province, West of Iran. Specimens' parasitological examination was performed by using the direct wet mount and formalin-ether concentration method. After DNA extraction from the samples, molecular analysis was done using PCR and LAMP techniques based on the Fasciola ribosomal intergenic spacer (IGS) sequence. Of 195 specimens of sheep, 11 specimens were identified as F. hepatica-positive infection by using microscopic, PCR and LAMP assays. Kappa agreement test results showed that there was a significant agreement between the results of microscopic examination diagnostic tests, PCR and LAMP (Kappa = 0.51-0.72 and p < .001). According to the results of chi-square comparisons between parasite prevalence applying different techniques and variables of age, sex breed, and type of drinking water, there was no significant relationship (p ≥ .05). However, most of the infected sheep with Fasciola were 3- to 4-year-old females, of the Lori breed and consumed tap water. In many endemic areas, successful prevention and treatment of fascioliasis in animals depend on rapid and accurate diagnosis. Based on the results of the Kappa agreement, the significant agreement among the results of the microscopic examination, PCR and LAMP indicates the accuracy and reliability of these tests in the diagnosis of F. hepatica in sheep. However, molecular methods, especially the LAMP technique, are suggested because of their higher sensitivity and reliability for the diagnosis of F. hepatica even under field conditions.

Development and evaluation of colloidal gold immunochromatography test strip for rapid diagnosis of nervous necrosis virus in golden grey mullet (Chelon aurata).

A lateral flow immunochromatography strip test, based on antibody-gold nanoparticles specific for nervous necrosis virus (NNV), was developed for rapid, on-site detection of the virus in fish stocks. A monoclonal antibody against NNV was conjugated with colloidal gold as the detector antibody. A rabbit anti-NNV polyclonal antibody and goat anti-mouse IgG antibody were blotted onto the nitrocellulose membrane as the capture antibodies on the test line and control line, respectively. The reaction could be seen by the eye within 15 min and did not cross-react with the other viruses tested. The detection limit of the strip was approximately 103 TCID50 /ml and had good stability after storage at 4°C for 8 months. When brains of 70 naturally infected golden grey mullet, Chelon aurata, were tested with the strip test, the diagnostic specificity and sensitivity of the test compared to real-time RT-PCR were 100% and 74%, respectively. Therefore, the one-step test strip developed here had high specificity, reproducibility, and stability. This, together with its simplicity to use and rapid detection, without the requirement of sophisticated equipment or specialized skills, makes the strip suitable for pond-side detection of NNV in farmed fish.

Comparison of three types of analyzers for urine protein-to-creatinine ratios in dogs.

BACKGROUND: Quantitation of urine protein is important in dogs with chronic kidney disease. Various analyzers are used to measure urine protein-to-creatinine ratios (UPCR). OBJECTIVES: This study aimed to compare the UPCR obtained by three types of analyzers (automated wet chemistry analyzer, in-house dry chemistry analyzer, and dipstick reading device) and investigate whether the differences could affect clinical decision process. METHODS: Urine samples were collected from 115 dogs. UPCR values were obtained using three analyzers. Bland-Altman and Passing Bablok tests were used to analyze agreement between the UPCR values. Urine samples were classified as normal or proteinuria based on the UPCR values obtained by each analyzer and concordance in the classification evaluated with Cohen's kappa coefficient. RESULTS: Passing and Bablok regression showed that there were proportional as well as constant difference between UPCR values obtained by a dipstick reading device and those obtained by the other analyzers. The concordance in the classification of proteinuria was very high (κ = 0.82) between the automated wet chemistry analyzer and in-house dry chemistry analyzer, while the dipstick reading device showed moderate concordance with the automated wet chemistry analyzer (κ = 0.52) and in-house dry chemistry analyzer (κ = 0.53). CONCLUSIONS: Although the urine dipstick test is simple and a widely used point-of-care test, our results indicate that UPCR values obtained by the dipstick test are not appropriate for clinical use. Inter-instrumental variability may affect clinical decision process based on UPCR values and should be emphasized in veterinary practice.

Prevalence and spatial distribution of Coxiella burnetii seropositivity in northern Australian beef cattle adjusted for diagnostic test uncertainty.

Q fever is a zoonotic disease caused by infection with Coxiella burnetii transmitted from animals including, but not limited to, cattle, sheep and goats. The infection in cattle is typically sub-clinical with some evidence suggesting associated reproductive loss. There is currently limited data on the true prevalence and distribution of coxiellosis in beef cattle across northern Australia. During this study, 2,012 sera samples from beef cattle managed on commercial farms located in Queensland and the Northern Territory were tested using an indirect immunofluorescent assay (IFA) for serological evidence of IgG antibodies against C. burnetii. Bayesian latent class models were used to estimate the true prevalence, adjusted for diagnostic test sensitivity and specificity and incorporating the hierarchical structure of the cattle within farms and regions. In this study, cattle in the Northern Territory had lower estimated true prevalence than cattle within most regions of Queensland with the exception of south-east Queensland. Results from this study have described the geographic distribution and estimated the true prevalence of antibodies to C. burnetii in a sample of extensively managed beef cattle located across the tropical grazing regions of northern Australia.

Molecular characterization and comparison of diagnostic methods for bovine respiratory viruses (BPIV-3, BRSV, BVDV, and BoHV-1) in field samples in northwestern Turkey.

The aim of this study was to evaluate the compatibility among virus isolation (VI), ELISA, and PCR for diagnosis of the major viral agents (BPIV-3, BRSV, BVDV, and BoHV-1) responsible for BRD in the field samples. For that purpose, a total of 193 samples (133 nasal swabs and 60 lung tissue samples) from cattle with respiratory signs in northwestern Turkey were examined. For VI, all the samples were inoculated at least 3 blind passages onto MDBK cell culture. In addition, the samples were tested by hemadsorption assay and RT-PCR for BPIV-3; nested RT-PCR for BRSV; immunoperoxidase monolayer assay, antigen-ELISA, and RT-PCR for BVDV; and antigen-ELISA and PCR for BoHV-1. The detected 1 (0.52%) BPIV-3 isolate was found to be in the genotype BPIV-3c. No BRSV isolate could be obtained, while 5 (2.59%) samples were evaluated positive in nested-RT PCR. The presence of BVDV antigen in 10 (5.18%) samples and the BVDV genome in 5 (2.59%) samples were detected, while non-cytopathogenic BVDV isolates were obtained only in 2 (1.04%) samples. The detected BVDV strains fell into the genetic clusters of BVDV-1a, -1f, and -1l. For detection of BoHV-1, although viral isolation and Ag-ELISA results were negative, presence of BoHV-1.1 genome was detected in 2 (1.04%) samples. By the results of VI, ELISA, and PCRs, 10.88% (21/193) of samples were found positive for the evaluated viruses. Depending on the obtained data, combined uses of the diagnostic methods were evaluated to be more reliable for routine diagnosis of bovine respiratory viruses.