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.S. Food and Drug Administration (FDA) and from the American Society for Veterinary Clinical Pathology (ASVCP). The LTC suggestions are closely aligned with those from the OIE and comply with version 2021-01 of the AAVLD Requirements for an Accredited Veterinary Medical Diagnostic Laboratory, although some LTC recommendations are more stringent and extend beyond the AAVLD requirements. LTC suggested guidelines are substantially different than the guidelines recently published by the U.S. FDA for validation and modification of regulated tests used for detection of pathogens in pet food and animal-derived products, such as dairy. Veterinary diagnostic laboratories that perform assays from the FDA Bacteriological Analytical Method (BAM) manual must be aware of the different standard.

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

Eurasian (EA)-origin H5N8 clade 2.3.4.4 avian influenza viruses were first detected in North America during December 2014. Subsequent reassortment with North American (AM) low-pathogenic wild-bird-origin avian influenza has generated at least two reassortants, including an EA/AM H5N1 from an apparently healthy wild green-winged teal, suggesting continued ongoing reassortment.

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.

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. The aim of this work is to report the detection of three avian influenza strains (H13N2, H5N9, H13N9) in gulls from Chile between 2007-2009, which nucleotide sequences showed highest similitudes to viruses detected in wild birds from North America. These results suggest a dissemination route for influenza viruses along the coasts of Americas. Migratory and synanthropic behaviors of birds included in this study support continued monitoring of avian influenza viruses isolated from wild birds in The Americas and the establishment of biosecurity practices in farms.

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. The full genome was sequenced from eight isolates as well as the haemagglutinin (HA) and neuraminidase (NA) gene segments of H7 and N9 virus subtypes for 108 isolates from North American wild birds between 1986 and 2012. Through maximum-likelihood and coalescent phylogenetic analyses, we identified the recent H7 and N9 IAV ancestors of the turkey-origin H7N9 IAVs, estimated the time and geographical origin of the ancestral viruses, and determined the relatedness between the 2009 and 2011 turkey-origin H7N9 IAVs. Analyses supported that the 2009 and 2011 viruses were distantly related genetically, suggesting that the two outbreaks arose from independent introduction events from wild birds. Our findings further supported that the 2011 MN turkey-origin H7N9 virus was closely related to H7N9 IAVs isolated in poultry in Nebraska during the same year. Although the precise origin of the wild-bird donor of the turkey-origin H7N9 IAVs could not be determined, our findings suggested that, for both the NA and HA gene segments, the MN turkey-origin H7N9 viruses were related to viruses circulating in wild birds between 2006 and 2011 in the Mississippi Flyway.

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. The basis of the HI test is inhibition of hemagglutination with subtype-specific antibodies. The HI assay is a relatively inexpensive procedure utilizing standard laboratory equipment, is less technical than molecular tests, and is easily completed within several hours. However when working with uncharacterized viruses or antibody subtypes the library of reference reagents required for identifying antigenically distinct influenza viruses and or antibody specificities from multiple lineages of a single hemagglutinin subtype requires extensive laboratory support for the production and optimization of reagents.

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. (Avian Dis 27:745-750, 1983). The micro-NI procedure reduces the quantity of reagents required, permits the antigenic classification of many isolates simultaneously, and eliminates spectrophotometric interpretation of results. Although, the macro-NI has been shown to be more sensitive than the micro-NI, the micro-NI test is very suitable for testing sera for the presence of NA antibodies and has proven to be a practical and rapid method for virus classification. This chapter provides an overview of the USDA validated micro-NI procedure for the identification of subtype-specific NA in AIV and antibodies.

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. The present study revealed that, for all of the validation factors evaluated, the candidate method performed at least as well and generally better than the current method. The candidate method was, therefore, deemed fit for the original intended purpose of the current method and rendered acceptable for use. A discussion of the case study is intended to further motivate consideration of the study designs chosen.

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. In 4-week-old SPF chickens, NDV-Belize-3/08 behaved as a typical velogenic viscerotropic NDV strain, causing severe necrohemorrhagic lesions in the lymphoid organs, with systemic virus distribution. Phylogenetic analysis of multiple NDV genotype V representatives revealed that genotype V can be divided into three subgenotypes, namely, Va, Vb, and Vc, and that all tested Belizean isolates belong to subgenotype Vb. Furthermore, these isolates are nearly identical to a 2007 isolate from Honduras and appear to have evolved separately from other contemporary viruses circulating in Mexico, clustering into a new clade within NDV subgenotype Vb.

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. Interviews were complemented by a survey on approaches to chronic disease management in each country. Finally two project teams (France and the Netherlands) conducted in-depth case studies on various aspects of chronic care evaluation. RESULTS: We identified three common challenges to evaluation of chronic disease management approaches: (1) a lack of evaluation culture and related shortage of capacity; (2) reluctance of payers or providers to engage in evaluation and (3) practical challenges around data and the heterogeity of IT infrastructure. The ability to evaluate chronic disease management interventions is influenced by contextual and cultural factors. CONCLUSIONS: This study contributes to our understanding of some of the most common underlying barriers to chronic care evaluation by highlighting the views and experiences of stakeholders and experts in six European countries. Overcoming the cultural, political and structural barriers to evaluation should be driven by payers and providers, for example by building in incentives such as feedback on performance, aligning financial incentives with programme objectives, collectively participating in designing an appropriate framework for evaluation, and making data use and accessibility consistent with data protection policies.

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. Using oro-pharyngeal and cloacal swabs collected from chickens experimentally exposed to the viruses we evaluated the effects of numerous aspects of sample collection and transport: 1) swab construction material (flocked nylon, non-flocked Dacron, or urethane foam), 2) transport media (brain heart infusion broth [BHI] or phosphate buffered saline [PBS]), 3) media volume (2 ml or 3.5 ml), 4) transporting the swab wet in the vial or removing the swab prior to transport, or transporting the swab dry with no media, and 5) single swabs versus pooling 5 or 11 swabs per vial. RESULTS: Using real-time RT-PCR (rRT-PCR), virus isolation (VI) and commercial antigen detection immunoassays for AIV we observed statistically significant differences and consistent trends with some elements of sample collection and transport; media, dry transport and swab construction. Conversely, the number of swabs pooled (1, 5 or 11) and whether the swab was removed prior to transport did not impact virus detection. Similarly, with NDV detection by both VI and rRT-PCR was not affected by the numbers of swabs collected in a single vial (1, 5 or 11). CONCLUSIONS: We observed that flocked and foam swabs were superior to non-flocked swabs, BHI media was better than PBS, and transporting swabs wet was better for virus recovery and detection than transporting them dry. There was no observable difference in detection whether the swab was removed prior to transport or left in the vial. Also, with both AIV and NDV, there was no observed difference in virus detection between pools of 1, 5 or 11 swabs.

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). The lack of any reported isolation of viruses related to this group since 1986 suggests that virulent viruses of this genotype may have evolved unnoticed for 22 years. The NDV-DR499-31/08 isolate had an intracerebral pathogenicity index (ICPI) score of 1.88, and sequencing of the fusion cleavage site identified multiple basic amino acids and a phenylalanine at position 117, indicating this isolate to be virulent. These results were further confirmed by a clinicopathological assessment in vivo. In 4-week-old chickens, NDV-DR499-31/08 behaved as a velogenic viscerotropic strain with systemic virus distribution and severe necrohemorrhagic lesions targeting mainly the intestine and the lymphoid organs. The clear phylogenetic relationship between the 2008, 1986, and 1947 ancestral viruses suggests that virulent NDV strains may have evolved in unknown reservoirs in the Caribbean and surrounding regions and underlines the importance of continued and improved epidemiological surveillance strategies to detect NDV in wild-bird species and commercial poultry.

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. A virulent Newcastle disease virus was also present in all three vials, but in low concentration. The virulence of the Newcastle disease virus was characterized by the intracerebral chicken pathogenicity index chicken inoculation assay but could not be determined by nucleotide sequence analysis from the virus isolated from embryonating chicken eggs. The intracerebral chicken pathogenicity index value for the isolated Newcastle disease virus was 1.55. Strains of Newcastle disease virus with intracerebral pathogenicity indexes significantly above 1.0 have been found to selectively kill many types of cancer cells while not affecting normal nonneoplastic cells and are considered to be a viable option for cancer treatment in humans by alternative medical researchers; however, the treatment is not approved for use in the United States by the Food and Drug Administration. Customs and Border Protection officials have been notified of an increased risk of Newcastle disease virus entering the United States for use as a nonapproved cancer treatment. Illegal importation of Newcastle disease vaccine for vaccination of backyard poultry is also a threat. This case report emphasizes the importance of conducting chicken inoculation for complete virus pathotyping and demonstrates the need for stringent security procedures at U.S. borders to detect known livestock pathogens that may be smuggled in for use in animal agriculture and reasons unrelated to animal agriculture.

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.

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. It has previously been shown to be unable to consistently detect all members of class I APMV-1. Diagnostic testing relies on rRT-PCR to quickly detect APMV-1 in wild birds, backyard flocks, live bird markets, commercial poultry, and for export testing. Limitations of the current USDA assay have raised concerns about the potential for some strains of APMV-1 to remain undetected by the primary screening assay. Mismatches in the probe were shown to cause a loss in template binding efficiency, resulting in lack of detection by the assay. Here, we describe the development and analytical validation of a new rRT-PCR assay designed to target a highly conserved region of the matrix gene across a wide range of APMV-1 strains. Limit of detection testing revealed a 3 log10 decrease in sensitivity for one low-virulence strain when compared to the USDA validated assay. Conversely, the assay showed increased sensitivity for a class I isolate and two virulent strains of APMV-1 that were not detected by the USDA-validated assay. The new assay also demonstrated a high degree of specificity by the lack of detection of 43 non-APMV-1 viruses.

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. Similarly, large differences were observed in the amino acid sequence of the fusion and hemagglutinin-neuraminidase proteins in comparison with all known NDV genotypes, suggesting the existence of an unknown reservoir for NDV. The work presented here represents the first complete genome sequence of NDV in the Dominican Republic.

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. Outbreaks of pandemic H1N1 infections in turkey breeder flocks were reported in Canada in 2009 and in the United States in 2010. Active surveillance of live bird markets in the United States led to the detection of H2, H3, H4, H5, H6, and H10 subtypes. Despite the fact that wild bird surveillance programs underwent contraction during this period in both Canada and the United States, H5 and H7 subtypes were still detected.

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. Analysis draws on results of a survey of 172 leaders of NHS organisations (primarily chief executives), 12 follow-up interviews, interviews with the SDU, and additional data and literature searches. A major conclusion is that almost all leaders consider sustainable development to be important for the NHS and that a focus on sustainability can most likely be aligned with delivering other corporate goals. Aligned incentives at all organisational levels and support for diversity are considered necessary to achieve sustainability, as well as relevant performance metrics. The main barrier is organisational culture.