Best practices for performance of real-time PCR assays in veterinary diagnostic laboratories.

The exquisite sensitivity of in vitro amplification assays such as real-time polymerase chain reaction (rtPCR) requires the establishment of thorough and robust laboratory practices. To this end, an American Association of Veterinary Laboratory Diagnosticians (AAVLD) committee of subject matter experts was convened to develop a set of best practices for performance of nucleic acid amplification assays. Consensus advice for the performance of preanalytical, analytical, and postanalytical steps is presented here, along with a review of supporting literature.

Suggested guidelines for validation of real-time PCR assays in veterinary diagnostic laboratories.

This consensus document presents the suggested guidelines developed by the Laboratory Technology Committee (LTC) of the American Association of Veterinary Laboratory Diagnosticians (AAVLD) for development, validation, and modification (methods comparability) of real-time PCR (rtPCR) assays. These suggested guidelines are presented with reference to the World Organisation for Animal Health (OIE) guidelines for validation of nucleic acid detection assays used in veterinary diagnostic laboratories. Additionally, our proposed practices are compared to the guidelines from the Foods Program Regulatory Subdivision of the U.

Novel Eurasian highly pathogenic avian influenza A H5 viruses in wild birds, Washington, USA, 2014.

Novel Eurasian lineage avian influenza A(H5N8) virus has spread rapidly and globally since January 2014. In December 2014, H5N8 and reassortant H5N2 viruses were detected in wild birds in Washington, USA, and subsequently in backyard birds. When they infect commercial poultry, these highly pathogenic viruses pose substantial trade issues.

Novel H5 Clade 2.3.4.4 Reassortant (H5N1) Virus from a Green-Winged Teal in Washington, USA.

Eurasian (EA)-origin H5N8 clade 2.3.4.

Avian Influenza in wild birds from Chile, 2007-2009.

Aquatic and migratory birds, the main reservoir hosts of avian influenza viruses including those with high pathogenic potential, are the wildlife species with the highest risk for viral dissemination across countries and continents. In 2002, the Chilean poultry industry was affected with a highly pathogenic avian influenza strain, which created economic loss and triggered the establishment of a surveillance program in wild birds. This effort consisted of periodic samplings of sick or suspicious animals found along the coast and analyses with standardized techniques for detection of influenza A virus.

H7N9 influenza A virus in turkeys in Minnesota.

Introductions of H7 influenza A virus (IAV) from wild birds into poultry have been documented worldwide, resulting in varying degrees of morbidity and mortality. H7 IAV infection in domestic poultry has served as a source of human infection and disease. We report the detection of H7N9 subtype IAVs in Minnesota (MN) turkey farms during 2009 and 2011.

Methods comparison.

The current report discusses the process in which a methods comparison study in the National Animal Health Laboratory Network is performed. Specific details are provided for designing and analyzing studies intended to evaluate analytical sensitivity, efficiency, analytical specificity, cross-contamination, repeatability, operator variability, and to compare the performance of methods using diagnostic samples. As an example, a case study is presented comparing the performance of a candidate reverse transcription polymerase chain reaction (RT-PCR) chemistry to the current RT-PCR chemistry in use when the assay was originally validated.

Neuraminidase-inhibition assay for the identification of influenza A virus neuraminidase virus subtype or neuraminidase antibody specificity.

The neuraminidase-inhibition (NI) assay is a laboratory procedure for the identification of the neuraminidase (NA) glycoprotein subtype in influenza viruses or the NA subtype specificity of antibodies to influenza virus. A serological procedure for subtyping the NA glycoprotein is critical for the identification and classification of avian influenza (AI) viruses. The macroprocedure was first described in 1961 by Aminoff and was later modified to a microtiter plate procedure (micro-NI) by Van Deusen et al.

Hemagglutination-inhibition assay for influenza virus subtype identification and the detection and quantitation of serum antibodies to influenza virus.

Hemagglutination-inhibition (HI) assay is a classical laboratory procedure for the classification or subtyping of hemagglutinating viruses. For influenza virus, HI assay is used to identify the hemagglutinin (HA) subtype of an unknown isolate or the HA subtype specificity of antibodies to influenza virus. Since the HI assay is quantitative it is frequently applied to evaluate the antigenic relationships between different influenza virus isolates of the same subtype.

Isolation of a virulent Newcastle disease virus from confiscated LaSota vaccine.

Vials of Newcastle disease vaccine labeled as LaSota were confiscated by the Arizona Division of Customs and Border Protection officials. Two different avian type 1 paramyxoviruses were isolated from all three vials of vaccine submitted to the National Veterinary Services Laboratories. The LaSota strain of avian paramyxovirus type 1 virus was isolated from all three vials and analyzed by nucleotide sequence analysis.

Separate evolution of virulent newcastle disease viruses from Mexico and Central America.

An outbreak of Newcastle disease (ND) in poultry was reported in Belize in 2008. The characteristics of three virulent Newcastle disease virus (NDV) isolates from this outbreak (NDV-Belize-3/08, NDV-Belize-4/08, and NDV-Belize-12/08) were assessed by genomic analysis and by clinicopathological characterization in specific-pathogen-free (SPF) chickens. The results showed that all three strains belong to NDV genotype V and are virulent, as assessed by the intracerebral pathogenicity index and the polybasic amino acid sequence at the fusion protein cleavage site.

Highly pathogenic avian influenza A(H7N3) virus in poultry workers, Mexico, 2012.

We identified 2 poultry workers with conjunctivitis caused by highly pathogenic avian influenza A(H7N3) viruses in Jalisco, Mexico. Genomic and antigenic analyses of 1 isolate indicated relatedness to poultry and wild bird subtype H7N3 viruses from North America. This isolate had a multibasic cleavage site that might have been derived from recombination with host rRNA.

Reported barriers to evaluation in chronic care: experiences in six European countries.

Introduction: The growing movement of innovative approaches to chronic disease management in Europe has not been matched by a corresponding effort to evaluate them. This paper discusses challenges to evaluation of chronic disease management as reported by experts in six European countries.

Methods: We conducted 42 semi-structured interviews with key informants from Austria, Denmark, France, Germany, The Netherlands and Spain involved in decision-making and implementation of chronic disease management approaches.

Optimal specimen collection and transport methods for the detection of avian influenza virus and Newcastle disease virus.

Background: Active and passive surveillance for avian influenza virus (AIV) and Newcastle disease virus (NDV) is widespread in commercial poultry worldwide, therefore optimization of sample collection and transport would be valuable to achieve the best sensitivity and specificity possible, and to develop the most accurate and efficient testing programs. A H7N2 low pathogenicity (LP) AIV strain was selected and used as an indicator virus because it is present in lower concentrations in swabbings and thus requires greater sensitivity for detection compared to highly pathogenic (HP) AIV. For similar reasons a mesogenic strain of NDV was selected.

Avian influenza in North America, 2009-2011.

All reports of avian influenza virus infections in poultry and isolations from wild bird species in Canada, the United States, and Mexico between 2009 and 2011 involved low pathogenic avian influenza. All three countries reported outbreaks of low pathogenic notifiable avian influenza in poultry during this period. The reports involved outbreaks of H5N2 among commercial turkeys in Canada in 2009 and 2010; outbreaks of H5N3 in turkeys in 2009, H5N2 in chickens in 2010, H7N3 in turkeys in 2011, and H7N9 in chickens, turkeys, geese, and guinea fowl in 2011 in the United States; and multiple outbreaks of H5N2 in chickens in Mexico in 2009, 2010, and 2011.

Highly divergent virulent isolates of Newcastle disease virus from the Dominican Republic are members of a new genotype that may have evolved unnoticed for over 2 decades.

A Newcastle disease virus (NDV) outbreak in chickens was reported in the Dominican Republic in 2008. The complete genome of this isolate, chicken/DominicanRepublic(JuanLopez)/499-31/2008 (NDV-DR499-31/08), and the fusion proteins of three other related viruses from the Dominican Republic and Mexico were sequenced and phylogenetically analyzed. Genetically, these four isolates were highly distinct from all other currently known isolates of NDV, and together, they fulfill the newly established criteria for inclusion as a novel genotype of NDV (genotype XVI).

Complete genome sequencing of a novel newcastle disease virus isolate circulating in layer chickens in the Dominican Republic.

Newcastle disease virus (NDV) was isolated from an outbreak in layer chickens in the Dominican Republic in 2008. Infections with this isolate led to a 100% apparent case fatality rate in birds. Complete genome sequencing revealed that the isolate does not belong to any of the previously described NDV genotypes.

An rRT-PCR assay to detect the matrix gene of a broad range of avian paramyxovirus serotype-1 strains.

The current U.S. Department of Agriculture (USDA)-validated real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay designed to detect the matrix gene of avian paramyxovirus serotype-1 (APMV-1) is the primary screening assay used in the United States.

Further Study on the Affordability of Alcoholic Beverages in the EU: A Focus on Excise Duty Pass-Through, On- and Off-Trade Sales, Price Promotions and Statutory Regulations.

Policies related to alcohol pricing, promotion and discounts provide opportunities to address harms associated with alcohol misuse. However, there are important gaps in information and knowledge about the regulations in place across parts of Europe and their impacts on consumer prices and locations of purchase. Using market data, we explored the overall scale and trend of price promotions and discounts in the off-premise (e.

Sustainable Development in the National Health Service (NHS): The Views and Values of NHS Leaders.

This article presents National Health Service (NHS) leaders' views of priorities and approaches regarding sustainable development in the NHS. It was produced in close collaboration with the United Kingdom (UK) NHS Sustainable Development Unit (SDU), and it represents the first systematic picture of leadership views in the NHS. It also provides a commentary on ways forward.

Recovery of H14 influenza A virus isolates from sea ducks in the Western Hemisphere.

In 2010, H14 influenza A viruses were recovered from clinically normal sea ducks in the United States. These are the first H14 isolates recovered in the Western Hemisphere and represent the only documented H14 influenza A viruses isolated since the original isolates were recovered from near the Caspian Sea during 1982.

Rapid PCR-based molecular pathotyping of H5 and H7 avian influenza viruses.

While the majority of avian influenza virus (AIV) subtypes are classified as low-pathogenicity avian influenza viruses (LPAIV), the H5 and H7 subtypes have the ability to mutate to highly pathogenic avian influenza viruses (HPAIV) in poultry and therefore are the etiological agents of notifiable AIV (NAIV). It is of great importance to distinguish HPAIV from LPAIV variants during H5/H7 outbreaks and surveillance. To this end, a novel and fast strategy for the molecular pathotyping of H5/H7 AIVs is presented.

How Health Systems Make Available Information on Service Providers: Experience in Seven Countries.

This article provides details on a report that reviews and discusses information systems reporting on the quality or performance of providers of healthcare ("quality information systems") in seven countries: Denmark, England, Germany, Italy, the Netherlands, Sweden and the United States. Data collection involves a review of the published and grey literature and is complemented by information provided by key informants in the selected countries using a detailed questionnaire. Quality information systems typically address a number of audiences, including patients (or respectively the general public before receiving services and becoming patients), commissioners, purchasers and regulators.

Evidence for a new avian paramyxovirus serotype 10 detected in rockhopper penguins from the Falkland Islands.

The biological, serological, and genomic characterization of a paramyxovirus recently isolated from rockhopper penguins (Eudyptes chrysocome) suggested that this virus represented a new avian paramyxovirus (APMV) group, APMV10. This penguin virus resembled other APMVs by electron microscopy; however, its viral hemagglutination (HA) activity was not inhibited by antisera against any of the nine defined APMV serotypes. In addition, antiserum generated against this penguin virus did not inhibit the HA of representative viruses of the other APMV serotypes.

Validation of a real-time reverse transcriptase-PCR assay for the detection of H7 avian influenza virus.

This report describes the validation of an avian influenza virus (AIV) H7 subtype-specific real-time reverse transcriptase-PCR (rRT-PCR) assay developed at the Southeast Poultry Research Laboratory (SEPRL) for the detection of H7 AI in North and South American wild aquatic birds and poultry. The validation was a collaborative effort by the SEPRL and the National Veterinary Services Laboratories. The 2008 H7 rRT-PCR assay detects 10(1) 50% embryo infectious doses per reaction, or 10(3)-10(4) copies of transcribed H7 RNA.