Validation of molecular-diagnostic techniques in the parasitological laboratory

Diagnostic laboratories today often operate according to standard quality management procedures such as ISO/IEC 17025. This requires that only validated methods are used. Validation procedures help to document that a particular protocol used by the accredited laboratory has a guaranteed performance in that particular laboratory. Several study designs exist for validation procedures. Computer programmes are available to help with the statistical analysis of validation results. The agreement beyond chance of results obtained in the protocol that is to be validated can be compared to those achieved in an already established test (agreement). For a method that is used under routine conditions or for epidemiological studies, it is necessary to assess the diagnostic sensitivity and diagnostic specificity of the technique. These parameters can be estimated by comparing the method that needs to be validated with an existing reliable method ('gold standard'). This is done by testing a standard set of well-documented samples using both techniques in parallel. Approaches using Bayes' theorem are used to perform gold standard-free validations. Many PCR-based methods are characterised by an excellent analytical sensitivity and are thus good candidates for diagnostic tools of the required diagnostic sensitivity. However, the high level of analytical sensitivity can also make molecular techniques susceptible to cross-contamination and carry-over problems leading to false-positive results. Moreover, the presence of inhibitors can cause false-negative results. After an initial validation, test performance needs to be continuously monitored, e.g. by using combined Shewhart-CUSUM control routines, and test results compared to those obtained by other laboratories (proficiency testing).     

The quality assurance of proficiency testing programs for animal disease diagnostic laboratories.

Laboratory data credibility has 3 major components: 1) valid methods, 2) proficiency testing (PT) to verify that the analyst can conduct the method and to compare results of other laboratories using the same method, and 3) third-party accreditation to verify that the laboratory is competent to conduct testing and that the method validation has been done within the environment and requirements of an effective quality-management system. Participation in external PT programs by a laboratory is strongly recommended in International Organization for Standardization/International Electrotechnical Commission International Standard 17025. Most laboratory accreditation bodies using this standard require that laboratories participate in such programs to be accredited. Internal PT is also recommended for each analyst. Benchmarking, or comparison between laboratories using PT or reference materials, is also recommended as part of the validation and evaluation of test methods. These requirements emphasize the need for proficiency test providers to demonstrate their competence. Requirements for competence are documented in national and international standards and guidelines, and accreditation is available for providers. This article discusses the activities and the components that are necessary and recommended for PT projects and programs for animal disease diagnostic testing. These are based on the requirements of the national and international standards, which address this subject, and on the experience of the author. The accreditation of external PT programs is also discussed. Organizations that accredit PT providers or that provide PT programs are listed. Existing references, guidelines, and standards that are relevant to PT in veterinary diagnostic laboratories are discussed.     

An internationally recognized quality assurance system for diagnostic parasitology in animal health and food safety, with example data on trichinellosis.

A quality assurance (QA) system was developed for diagnostic parasitology and implemented for several diagnostic assays including fecal flotation and sedimentation assays, trichomonad culture assay, and the testing of pork and horse meat for Trichinella to facilitate consistently reliable results. The system consisted of a validated test method, procedures to confirm laboratory capability, and protocols for documentation, reporting, and monitoring. Specific system components included a quality assurance manual, training program, proficiency panels, inter-laboratory check-sample exchange program, assay critical control points, controls, and audits. The quality assurance system of the diagnostic laboratory was audited according to ISO/IEC Standard 17025 by an international third party accrediting body and accredited as a testing laboratory for the specific parasitology tests. Test results generated from the laboratory were reliable and scientifically defensible according to the defined parameters of the tests and were therefore valid for a variety of purposes, including food safety, international trade, and declaration of disease status in an animal, herd, farm, or region. The system was applicable to various test methods for the detection of parasites in feces or other samples, and a digestion test system developed for Trichinella was used as an example. A modified tissue digestion assay was developed, validated, and implemented by the Canadian Food Inspection Agency's Centre for Animal Parasitology for efficiency and quality assurance. The details of the method were properly documented for routine testing and consisted of a homogenization process, an incubation at 45+/-2 degrees C, and two sequential sedimentations in separatory funnels to concentrate and clarify final aliquots for microscopic examination. To facilitate consistently reliable test results, 14 critical control points were identified and monitored, analysts were certified, and the test system verified through the use of validation data, proficiency samples, and training modules.     

超高频电子耳标物理层及标签识别层测试方法研究

基于RFID技术的电子耳标追溯体系可进行动物出生后、屠宰前的精准定位,实现畜产品的可追溯性管理。超高频电子耳标测试系统具有较远的读取距离及较高的传输速率,广泛应用于畜产品的可追溯性领域。该类标签的存储地址依据空中接口协议标准ISO18000-63定义,测试项目依据18047-6测试方法进行。对测试项目的测试方法和判定依据进行了详细介绍,并对链接时间T1进行10次测试,计算其测量不确定度,测量结果显示符合标准要求。     

Validation of 2 commercial Neospora caninum antibody enzyme linked immunosorbent assays

This is a validation study of 2 commercially available enzyme linked immunosorbent assays (ELISA) for the detection of antibodies against Neospora caninum in bovine serum. The results of the reference sera (n = 30) and field sera from an infected beef herd (n = 150) were tested by both ELISAs and the results were compared statistically. When the immunoblotting results of the reference bovine sera were compared to the ELISA results, the same identity score (96.67%) and kappa values (K) (0.93) were obtained for both ELISAs. The sensitivity and specificity values for the IDEXX test were 100% and 93.33% respectively. For the Biovet test 93.33% and 100% were obtained. The corresponding positive (PV+) and negative predictive (PV−) values for the 2 assays were 93.75% and 100% (IDEXX), and 100% and 93.75% (Biovet). In the 2nd study, competitive inhibition ELISA (c-ELISA) results on bovine sera from an infected herd were compared to the 2 sets of ELISA results. The identity scores of the 2 ELISAs were 98% (IDEXX) and 97.33% (Biovet). The K values calculated were 0.96 (IDEXX) and 0.95 (Biovet). For the IDEXX test the sensitivity and specificity were 97.56% and 98.53%, whereas for the Biovet assay 95.12% and 100% were recorded, respectively. The corresponding PV+ and PV− values were 98.77% and 97.1% (IDEXX), and 100% and 94.44% (Biovet). Our validation results showed that the 2 ELISAs worked equally well and there was no statistically significant difference between the performance of the 2 tests. Both tests showed high reproducibility, repeatability and substantial agreement with results from 2 other laboratories. A quality assurance based on the requirement of the ISO/IEC 17025 standards has been adopted throughout this project for test validation procedures.     

The detection of anthelmintic resistance in nematodes of veterinary importance

Before revised World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines on the detection of anthelmintic resistance can be produced, validation of modified and new methods is required in laboratories in different parts of the world. There is a great need for improved methods of detection of anthelmintic resistance particularly for the detection of macrocyclic lactone resistance and for the detection of resistant nematodes in cattle. Therefore, revised and new methods are provided here for the detection of anthelmintic resistance in nematodes of ruminants, horses and pigs as a basis for discussion and with the purpose that they are evaluated internationally to establish whether they could in the future be recommended by the WAAVP. The interpretation of the faecal egg count reduction test has been modified and suggestions given on its use with persistent anthelmintics and continuous release devices. An egg hatch test for benzimidazole (BZ) resistance is described. A microagar larval development test for the detection of benzimidazole and levamisole resistance provides third stage larvae for the identification of resistant worms. The sensitivity of these two tests can be increased by using discriminating doses rather than LD(50) values. Details are given of a PCR based test for the analysis of benzimidazole resistance in strongyles of sheep and goats, horses and cattle. Although promising for ruminant trichostrongyles, quantitative determination of gene frequency using real time PCR requires further development before PCR tests will be used in the field. Apart from faecal egg count reduction tests there are currently no satisfactory tests for macrocylic lactone resistance despite the great importance of this subject. Except for treatment and slaughter trials there are no validated tests for fasciolicide resistance or for the detection of resistance in cestodes.     

Trichinella diagnostics and control: Mandatory and best practices for ensuring food safety

Because of its role in human disease, there are increasing global requirements for reliable diagnostic and control methods for Trichinella in food animals to ensure meat safety and to facilitate trade. Consequently, there is a need for standardization of methods, programs, and best practices used in the control of Trichinella and trichinellosis. This review article describes the biology and epidemiology of Trichinella, and describes recommended test methods as well as modified and optimized procedures that are used in meat inspection programs. The use of ELISA for monitoring animals for infection in various porcine and equine pre- and post-slaughter programs, including farm or herd certification programs is also discussed. A brief review of the effectiveness of meat processing methods, such as freezing, cooking and preserving is provided. The importance of proper quality assurance and its application in all aspects of a Trichinella diagnostic system is emphasized. It includes the use of international quality standards, test validation and standardization, critical control points, laboratory accreditation, certification of analysts and proficiency testing. Also described, are the roles and locations of international and regional reference laboratories for trichinellosis where expert advice and support on research and diagnostics are available.     

射频识别技术在牲畜电子耳标检测上的运用

牲畜电子耳标是射频识别技术在动物身份识别上的典型运用,在我国一些省份进行了试点佩戴合和推行。本文简述射频识别的原理,将超高频电子耳标引用的国际标准ISO/IEC18000-6C和国家标准GB/T29768进行对比,对其中频率范围、编码方式等8个技术指标进行说明,对检测中的频点选择、链接时间T1的计算和识读距离等项目进行解释。以期对超高频电子耳标的检测发挥指导作用。     

ASVCP quality assurance guidelines: control of preanalytical, analytical, and postanalytical factors for urinalysis, cytology, and clinical chemistry in veterinary laboratories

In December 2009, the American Society for Veterinary Clinical Pathology (ASVCP) Quality Assurance and Laboratory Standards committee published the updated and peer-reviewed ASVCP Quality Assurance Guidelines on the Society's website. These guidelines are intended for use by veterinary diagnostic laboratories and veterinary research laboratories that are not covered by the US Food and Drug Administration Good Laboratory Practice standards (Code of Federal Regulations Title 21, Chapter 58). The guidelines have been divided into 3 reports: (1) general analytical factors for veterinary laboratory performance and comparisons; (2) hematology, hemostasis, and crossmatching; and (3) clinical chemistry, cytology, and urinalysis. This particular report is one of 3 reports and documents recommendations for control of preanalytical, analytical, and postanalytical factors related to urinalysis, cytology, and clinical chemistry in veterinary laboratories and is adapted from sections 1.1 and 2.2 (clinical chemistry), 1.3 and 2.5 (urinalysis), 1.4 and 2.6 (cytology), and 3 (postanalytical factors important in veterinary clinical pathology) of these guidelines. These guidelines are not intended to be all-inclusive; rather, they provide minimal guidelines for quality assurance and quality control for veterinary laboratory testing and a basis for laboratories to assess their current practices, determine areas for improvement, and guide continuing professional development and education efforts.     

Developments in international standardization

Trade in animals and animal products has reached global proportions and so too has the threat of infectious diseases of veterinary importance. The Manual of Standards for Diagnostic Tests and Vaccines, published by the Office International des Epizooties (OIE), contains chapters on infectious diseases that may cause various degrees of socio-economic, public health, and/or zoo-sanitary consequence. These chapters cover the major diseases of cattle, sheep, goats, horses, pigs, poultry, lagomorphs and bees. A number of factors are considered when qualifying animals and animal products for international trade including epidemiological, clinical and testing parameters. Of particular note and relevance is a strong international movement to standardize the test methods and reference reagents in order to promote harmonization of testing and facilitation of trade. There is message here that is directed to those of us involved in the development and application of test methods for infectious disease diagnosis. Serological test methods have been and still remain the mainstay of diagnostic methods prescribed for trade. More than ever, there is a need to observe and apply international guidelines for the development and validation of serological test methods. There is also a need to develop international standard reagents for use in the calibration of test methods and the production of national and working standards. In the future, veterinary diagnostic testing laboratories involved in trade may also require a form of international accreditation unique to their specialty. This presentation describes the current developments in international standardization of test methods and reference reagents.     

ASVCP reference interval guidelines: determination of de novo reference intervals in veterinary species and other related topics

Reference intervals (RI) are an integral component of laboratory diagnostic testing and clinical decision‐making and represent estimated distributions of reference values (RV) from healthy populations of comparable individuals. Because decisions to pursue diagnoses or initiate treatment are often based on values falling outside RI, the collection and analysis of RV should be approached with diligence. This report is a condensation of the ASVCP 2011 consensus guidelines for determination of de novo RI in veterinary species, which mirror the 2008 Clinical Laboratory and Standards Institute (CLSI) recommendations, but with language and examples specific to veterinary species. Newer topics include robust methods for calculating RI from small sample sizes and procedures for outlier detection adapted to data quality. Because collecting sufficient reference samples is challenging, this document also provides recommendations for determining multicenter RI and for transference and validation of RI from other sources (eg, manufacturers). Advice for use and interpretation of subject‐based RI is included, as these RI are an alternative to population‐based RI when sample size or inter‐individual variation is high. Finally, generation of decision limits, which distinguish between populations according to a predefined query (eg, diseased or non‐diseased), is described. Adoption of these guidelines by the entire veterinary community will improve communication and dissemination of expected clinical laboratory values in a variety of animal species and will provide a template for publications on RI. This and other reports from the Quality Assurance and Laboratory Standards (QALS) committee are intended to promote quality laboratory practices in laboratories serving both clinical and research veterinarians.     

ASVCP quality assurance guidelines: control of preanalytical and analytical factors for hematology for mammalian and nonmammalian species, hemostasis, and crossmatching in veterinary laboratories

In December 2009, the American Society for Veterinary Clinical Pathology (ASVCP) Quality Assurance and Laboratory Standards committee published the updated and peer-reviewed ASVCP Quality Assurance Guidelines on the Society's website. These guidelines are intended for use by veterinary diagnostic laboratories and veterinary research laboratories that are not covered by the US Food and Drug Administration Good Laboratory Practice standards (Code of Federal Regulations Title 21, Chapter 58). The guidelines have been divided into 3 reports: (1) general analytical factors for veterinary laboratory performance and comparisons; (2) hematology, hemostasis, and crossmatching; and (3) clinical chemistry, cytology, and urinalysis. This particular report is one of 3 reports and provides recommendations for control of preanalytical and analytical factors related to hematology for mammalian and nonmammalian species, hemostasis testing, and crossmatching and is adapted from sections 1.1 and 2.3 (mammalian hematology), 1.2 and 2.4 (nonmammalian hematology), 1.5 and 2.7 (hemostasis testing), and 1.6 and 2.8 (crossmatching) of the complete guidelines. These guidelines are not intended to be all-inclusive; rather, they provide minimal guidelines for quality assurance and quality control for veterinary laboratory testing and a basis for laboratories to assess their current practices, determine areas for improvement, and guide continuing professional development and education efforts.     

狂犬病毒G蛋白原核表达及间接ELISA检测方法的建立

目前狂犬病疫苗的效力检验采用NIH法,需要使用狂犬病毒强毒株进行攻毒试验,具有一定的生物安全风险。为建立检测小鼠血清抗体效价的间接ELISA方法,代替NIH法中的脑内攻毒试验,扩增狂犬病毒（RV）G蛋白基因,并将其克隆至大肠杆菌pET-32a载体上进行表达。以该蛋白作包被抗原,摸索试验条件,建立检测小鼠血清抗体效价的间接ELISA方法。使用此方法与国际公认的荧光抗体病毒中和试验（FAVN）法比较,两者检测结果曲线相关系数为0.986,表明两者相关性较好,但ELSIA方法更加快捷、简便。本试验建立的间接ELISA方法可用于检测小鼠血清中狂犬病抗体,为狂犬病疫苗效力检验替代方法的建立提供了基础。     

ASVCP quality assurance guidelines: control of general analytical factors in veterinary laboratories

Owing to lack of governmental regulation of veterinary laboratory performance, veterinarians ideally should demonstrate a commitment to self-monitoring and regulation of laboratory performance from within the profession. In response to member concerns about quality management in veterinary laboratories, the American Society for Veterinary Clinical Pathology (ASVCP) formed a Quality Assurance and Laboratory Standards (QAS) committee in 1996. This committee recently published updated and peer-reviewed Quality Assurance Guidelines on the ASVCP website. The Quality Assurance Guidelines are intended for use by veterinary diagnostic laboratories and veterinary research laboratories that are not covered by the US Food and Drug Administration Good Laboratory Practice standards (Code of Federal Regulations Title 21, Chapter 58). The guidelines have been divided into 3 reports on 1) general analytic factors for veterinary laboratory performance and comparisons, 2) hematology and hemostasis, and 3) clinical chemistry, endocrine assessment, and urinalysis. This report documents recommendations for control of general analytical factors within veterinary clinical laboratories and is based on section 2.1 (Analytical Factors Important In Veterinary Clinical Pathology, General) of the newly revised ASVCP QAS Guidelines. These guidelines are not intended to be all-inclusive; rather, they provide minimum guidelines for quality assurance and quality control for veterinary laboratory testing. It is hoped that these guidelines will provide a basis for laboratories to assess their current practices, determine areas for improvement, and guide continuing professional development and education efforts.     

Reference values: a review

Abstract: Reference values are used to describe the dispersion of variables in healthy individuals. They are usually reported as population‐based reference intervals (RIs) comprising 95% of the healthy population. International recommendations state the preferred method as a priori nonparametric determination from at least 120 reference individuals, but acceptable alternative methods include transference or validation from previously established RIs. The most critical steps in the determination of reference values are the selection of reference individuals based on extensively documented inclusion and exclusion criteria and the use of quality‐controlled analytical procedures. When only small numbers of values are available, RIs can be estimated by new methods, but reference limits thus obtained may be highly imprecise. These recommendations are a challenge in veterinary clinical pathology, especially when only small numbers of reference individuals are available.     

Flow cytometry as a method for the evaluation of raw material, product and process in the dairy industry

Producing dairy products which are safe for consumers requires the constant monitoring of the microbiological quality of raw material, the production process itself and the end product. Traditional methods, still a "gold standard", require a specialized laboratory working on recognized and validated methods. Obtaining results is time- and labor-consuming and do not allow rapid evaluation. Hence, there is a need for a rapid, precise method enabling the real-time monitoring of microbiological quality, and flow cytometry serves this function well. It is based on labeling cells suspended in a solution with fluorescent dyes and pumping them into a measurement zone where they are exposed to a precisely focused laser beam. This paper is aimed at presenting the possibilities of applying flow cytometry in the dairy industry.     

Comparison of DNA isolation methods and detection of Salmonella spp. from animal faeces and dust using invA real-time PCR

There is a strong interest to reduce the expenditure for the detection of Salmonella spp. from animal faeces and environmental samples from primary production according to ISO 6579:2002 Annex D by including a rapid and effective method to detect Salmonella spp. already after pre-enrichment in BPW. It has been shown that real-time PCR methods are very effective to detect Salmonella organisms after pre-enrichment of foods. However, materials from primary animal production compose of much higher amounts of substances which might inhibit the sensitivity of real-time PCR. Different techniques of DNA isolation after pre-enrichment of artificially inoculated bovine faecal material were used to compare their detection limit and detection probability using an invA 5' nuclease real-time PCR approach. A detection probability of 100% was shown at 10(5) cfu/ml using the QIAamp DNA Stool Mini Kit (Qiagen, Germany), at 10(4) cfu/ml using the High Pure PCR Template Preparation Kit (Roche, Germany) and at 10(3) cfu/ml using thermal cell lysis or an in-house lab protocol, respectively. In comparison DNA isolation by thermal cell lysis revealed a very good detection limit, low costs and almost no risks of contamination. Furthermore, caecal contents from pigs were analysed by ISO 6579:2002 Annex D and the invA real-time PCR using thermal cell lysis for DNA extraction. As a result neither false positive nor false negative findings were obtained. Inclusion of the real-time PCR after pre-enrichment of samples in BPW followed by bacterial detection of Salmonella only with samples positive with real-time PCR might be a valuable tool to fulfil the international standard of ISO 6579:2002 Annex D but also to diminish the expenditures. However, it must be stated that the modification of an international standard method and its use in routine diagnostic requires the validation and registration of national and/or international competent authorities.     

A review of methods for assessment of the rate of gastric emptying in the dog and cat: 1898-2002

Gastric emptying is the process by which food is delivered to the small intestine at a rate and in a form that optimizes intestinal absorption of nutrients. The rate of gastric emptying is subject to alteration by physiological, pharmacological, and pathological conditions. Gastric emptying of solids is of greater clinical significance because disordered gastric emptying rarely is detectable in the liquid phase. Imaging techniques have the disadvantage of requiring restraint of the animal and access to expensive equipment. Radiographic methods require administration of test meals that are not similar to food. Scintigraphy is the gold standard method for assessment of gastric emptying but requires administration of a radioisotope. Magnetic resonance imaging has not yet been applied for assessment of gastric emptying in small animals. Ultrasonography is a potentially useful, but subjective, method for assessment of gastric emptying in dogs. Gastric tracer methods require insertion of gastric or intestinal cannulae and are rarely applied outside of the research laboratory. The paracetamol absorption test has been applied for assessment of liquid phase gastric emptying in the dog, but requires IV cannulation. The gastric emptying breath test is a noninvasive method for assessment of gastric emptying that has been applied in dogs and cats. This method can be carried out away from the veterinary hospital, but the effects of physiological and pathological abnormalities on the test are not known. Advances in technology will facilitate the development of reliable methods for assessment of gastric emptying in small animals.