Chromosome microarray proficiency testing and analysis of quality metric data trends through an external quality assessment program for Australasian laboratories.

Chromosome microarrays are an essential tool for investigation of copy number changes in children with congenital anomalies and intellectual deficit. Attempts to standardise microarray testing have focused on establishing technical and clinical quality criteria, however external quality assessment programs are still needed. We report on a microarray proficiency testing program for Australasian laboratories. Quality metrics evaluated included analytical accuracy, result interpretation, report completeness, and laboratory performance data: sample numbers, success and abnormality rate and reporting times. Between 2009 and 2014 nine samples were dispatched with variable results for analytical accuracy (30-100%), correct interpretation (32-96%), and report completeness (30-92%). Laboratory performance data (2007-2014) showed an overall mean success rate of 99.2% and abnormality rate of 23.6%. Reporting times decreased from >90 days to <30 days for normal results and from >102 days to <35 days for abnormal results. Data trends showed a positive correlation with improvement for all these quality metrics, however only 'report completeness' and reporting times reached statistical significance. Whether the overall improvement in laboratory performance was due to participation in this program, or from accumulated laboratory experience over time, is not clear. Either way, the outcome is likely to assist referring clinicians and improve patient care.

A survey of wild marine fish identifies a potential origin of an outbreak of viral haemorrhagic septicaemia in wrasse, Labridae, used as cleaner fish on marine Atlantic salmon, Salmo salar L., farms.

Viral haemorrhagic septicaemia virus (VHSV) was isolated from five species of wrasse (Labridae) used as biological controls for parasitic sea lice predominantly, Lepeophtheirus salmonis (Krøyer, 1837), on marine Atlantic salmon, Salmo salar L., farms in Shetland. As part of the epidemiological investigation, 1400 wild marine fish were caught and screened in pools of 10 for VHSV using virus isolation. Eleven pools (8%) were confirmed VHSV positive from: grey gurnard, Eutrigla gurnardus L.; Atlantic herring, Clupea harengus L.; Norway pout, Trisopterus esmarkii (Nilsson); plaice, Pleuronectes platessa L.; sprat, Sprattus sprattus L. and whiting, Merlangius merlangus L. The isolation of VHSV from grey gurnard is the first documented report in this species. Nucleic acid sequencing of the partial nucleocapsid (N) and glycoprotein (G) genes was carried out for viral characterization. Sequence analysis confirmed that all wild isolates were genotype III the same as the wrasse and there was a close genetic similarity between the isolates from wild fish and wrasse on the farms. Infection from these local wild marine fish is the most likely source of VHSV isolated from wrasse on the fish farms.

Development and validation of a range of endogenous controls to support the implementation of practical Taqman real-time PCR-based surveillance for fish diseases within aquaculture.

The use of Taqman real-time PCR-based technology has recently become more frequent in the detection of pathogens in the aquaculture industry. This interest has necessitated the development of robust and reliable pathogen-detection assays. The development of a range of endogenous control assays to be run alongside these diagnostic assays works to further increase confidence in the latter. This study describes the design of a range of endogenous control assays based on the elongation factor 1-α (EF1-α) gene specific to a range of fish species including Atlantic salmon, Salmo salar; rainbow trout, Oncorhynchus mykiss; brown trout, Salmo trutta; cod, Gadus morhua; haddock, Melanogrammus aeglefinus; saithe, Pollachius virens; whiting, Merlangius merlangus; Norway pout, Trisopterus esmarkii; carp (family Cyprinidae), roach, Rutilus rutilus; European eel, Anguilla anguilla; and herring, Clupea harengus, as well as a number of fish cell lines. Evidence is provided of the validation of these assays for specific species, a range of tissue types and cell lines as well as an example of the potential uses of these assays.

The variant methylenetetrahydrofolate reductase c.1298A>C (p.E429A) is associated with multiple sclerosis in a German case-control study.

Multiple sclerosis (MS) is an inflammatory demyelinating autoimmune disease of the central nervous system. We investigated the association of two missense variants of the MTHFR gene, i.e. MTHFR c.677C>T (p.A222V) and c.1298A>C (p.E429A), in 138 patients with clinically definite multiple sclerosis of relapsing-remitting course and 138 age- and gender-matched healthy controls. No significant differences were found in the frequency of the MTHFR c.677C>T polymorphism between MS patients and healthy controls. However, the genotype frequencies of the missense variant MTHFR c.1298A>C were significantly different between patients (AA/AC/CC: 0.34/0.55/0.11) and controls (0.52/0.36/0.12; Pearson's chi(2)=11.1; p=0.004). These results suggest that homozygosity for the A allele of MTHFR c.1298A>C may be protective against the incidence of MS. If confirmed in an independent study sample, the underlying mechanisms should be investigated, which may lead to novel insights in biochemical factors influencing the aetiology and pathophysiology of MS.

Genetic analysis of infectious pancreatic necrosis virus from Scotland.

Infectious pancreatic necrosis (IPN) is a highly contagious disease of young salmonid fish, and is one of the most serious economic diseases in aquaculture. In Scotland, an increase in IPN virus (IPNV) outbreaks in seawater Atlantic salmon, Salmo salar, has been reported in recent years. The aim of this study was to analyse the VP2 gene from recent IPNV isolates from Scotland, to determine whether there are epidemiological links between IPNV isolates from farms (13), wild fish (17) and the environment (6) in order to investigate potential wild and farmed fish interactions. Comparison of the nucleotide sequence of the VP2 gene revealed that 34 of 36 isolates were 97.1-100% similar and the deduced amino acid sequences showed 97-100% identity. Two isolates from wild fish exhibited the most divergence at 85-87.3% similarity to the other isolates at the nucleotide level and 88.2-90.8% identity at the deduced amino acid level. Phylogenetic analyses revealed that 34 of 36 of the isolates from Scotland were genetically closely related to the A2 (Sp) serotype of IPNV. The two wild isolates from seatrout, Salmo trutta, and flounder, Platichthys flesus, were most closely related to the European A5 (Te) serotype. This study represents a comprehensive IPNV phylogenetic study that indicates that there are closely related or identical isolates in circulation in the marine environment, which adds evidence that disease interactions between wild and farmed fish may occur. This type of analysis is a useful tool in the management and control of fish diseases because it can assist in the identification of epidemiological links and highlight potential risks to aquaculture.

Detection of infectious pancreatic necrosis virus (IPNV) from the environment in the vicinity of IPNV-infected Atlantic salmon farms in Scotland.

Infectious pancreatic necrosis virus (IPNV) has been isolated from mussels, sediment and surface water in the vicinity of clinically infected salmon farms, at shore bases supplying the farms and for several hundred metres distance from farms in the direction of current flow. There was evidence of decreasing prevalence of IPNV in mussels from Shetland once IPN outbreaks subsided, indicating they are an unlikely source of re-infection on farms. There was little evidence of persistence in the environment, although conclusions were complicated by the presence of IPNV on neighbouring farms 1 year after the outbreak.

Infectious pancreatic necrosis virus in Atlantic salmon, Salmo salar L., post-smolts in the Shetland Isles, Scotland: virus identification, histopathology, immunohistochemistry and genetic comparison with Scottish mainland isolates.

During mid-June 1999 peak mortalities of 11% of the total stock per week were seen at a sea cage site of Atlantic salmon, Salmo salar L., post-smolts in the Shetland Isles, Scotland. Virus was isolated on chinook salmon embryo (CHSE) cells in a standard diagnostic test and infectious pancreatic necrosis virus (IPNV) identified by enzyme-linked immunosorbent assay. IPNV was confirmed as serogroup A by a cell immunofluorescent antibody test using the cross-reactive monoclonal antibody AS-1. Four weeks after the main outbreak, virus titres in surviving moribund fish were assayed at >10(10) TCID50 g(-1) kidney. Histopathology of moribund fish was characterized by pancreatic acinar cell necrosis and a marked catarrhal enteritis of the intestinal mucosa. In the liver, necrosis, leucocytic infiltration and a generalized cell vacuolation were noted. IPNV-specific immunostaining was demonstrated in pancreas, liver, heart, gill and kidney tissue. The nucleotide sequence of the coding region of segment A was determined from the Shetland isolate. A 1180 bp fragment of the VP2 gene of this isolate was compared with a 1979 reference isolate from mainland Scottish Atlantic salmon, La/79 and another more recent mainland isolate, 432/00. Both A2 isolates were derived from carrier fish without signs of IPN and serotyped by a plaque neutralization test. The Shetland isolate shows a different nucleotide and amino acid sequence compared with the two isolates from carrier fish. These latter isolates showed identical amino acid sequences in the fragment examined, despite the 21 years separating the isolations. Sequence comparisons with other A2 (Sp) isolates on the database confirm all three Scottish isolates are A2 (Sp).

Genotyping of marine viral haemorrhagic septicaemia virus isolated from the Flemish Cap by nucleotide sequence analysis and restriction fragment length polymorphism patterns.

A total of 14 viral haemorrhagic septicaemia virus (VHSV) isolates obtained from Greenland halibut Reinhardtius hippoglossoides caught at the Flemish Cap, a fishing ground in the North Atlantic Ocean near Newfoundland, were characterised using restriction fragment length polymorphism (RFLP) and nucleotide sequence analysis. RFLP analysis was performed on a 1259 bp fragment of the glycoprotein (G) gene, and a 305 nucleotide region within the nucleoprotein (N) gene was used for sequence analysis. Representative strains of the 4 established genotypes were employed for comparative purposes. Sequencing analysis indicated that the Flemish cap isolates grouped in Genotype 3, which also includes isolates from wild fish caught in the North Sea and coastal waters of the UK and Ireland, isolates derived from outbreaks of VHS in turbot farms in the British Isles, and an isolate from European eel Anguilla anguilla caught in northern France. Characterisation using RFLPs resulted in the development of a simple and reliable method of typing VHSV at the genotype level using a 2-step restriction analysis (2-SRA) assay.

Genetic population structure of marine viral haemorrhagic septicaemia virus (VHSV).

The nucleotide sequences of a specific region of the nucleoprotein gene were compared in order to investigate the genetic population structure of marine viral haemorrhagic septicaemia virus (VHSV). Analysis of the sequence from 128 isolates of diverse geographic and host origin renders this the most comprehensive molecular epidemiological study of marine VHSV conducted to date. Phylogenetic analysis of nucleoprotein gene sequences confirmed the existence of the 4 major genotypes previously identified based on N- and subsequent G-gene based analyses. The range of Genotype I included subgroups of isolates associated with rainbow trout aquaculture (Genotype Ia) and those from the Baltic marine environment (Genotype Ib) to emphasise the relatively close genetic relationship between these isolates. The existence of an additional genotype circulating within the Baltic Sea (Genotype II) was also confirmed. Genotype III included marine isolates from around the British Isles in addition to those associated with turbot mariculture, highlighting a continued risk to the development of this industry. Genotype IV consisted of isolates from the marine environment in North America. Taken together, these findings suggest a marine origin of VHSV in rainbow trout aquaculture. The implications of these findings with respect to the future control of VHSV are discussed. The capacity for molecular phylogenetic analysis to resolve complex epidemiological problems is also demonstrated and its likely future importance to disease management issues highlighted.

An evaluation of current diagnostic tests for the detection of infectious salmon anaemia virus (ISAV) following experimental water-borne infection of Atlantic salmon, Salmo salar L.

Four commonly used diagnostic tests [reverse transcription polymerase chain reaction (RT-PCR), indirect fluorescent antibody test (IFAT), virus culture and light microscopy] were evaluated for their ability to detect infectious salmon anaemia virus (ISAV) or tissue pathology following experimental infection of Atlantic salmon. Fish were infected with ISAV by water-borne exposure which mimics the route of natural infection. Forty-five per cent of pre-clinical fish tested yielded positive results by RT-PCR for at least one of the organs tested (kidney, heart, gill, liver, blood). No significant difference was detected between organs in the number or time of first occurrence of positive result. Virus culture identified a total of 14% of pre-clinical fish as ISAV-infected. The presence of ISAV in heart tissue was particularly notable (13% of fish sampled) as was the inability to culture virus from spleen tissue. In the case of IFAT, 15% of fish sampled were positive, although tissue other than kidney proved unsuitable for use in this method. Only limited ISAV-specific pathology was detectable by histological examination of fish prior to the onset of clinical disease. These findings reveal important information regarding the optimal choice of both tissue sample and diagnostic test for the routine diagnosis of ISAV.

Treating patients with colorectal cancer in rural and urban areas: a qualitative study of the patients' perspective.

OBJECTIVES: The aim of the present study was to explore the perspectives of patients receiving treatment for colorectal cancer and compare priorities and attitudes in rural and urban areas. METHOD: A qualitative study was carried out involving four focus groups in the Aberdeen and Northern Centre for Haematology, Oncology and Radiotherapy. The sample comprised 22 patients at various stages of treatment for colorectal cancer and 10 of their relatives from different locations of Northeast Scotland and Shetland. The main themes generated by participants were identified, and similarities and differences between urban and rural patients were noted within these themes. RESULTS: Components of care that were important to rural and urban patients were speed of referral to specialists for treatment and issues of communication including test results and delivery of bad news. Tensions were perceived at the interface of primary and secondary care and these were blamed for delays in referral and communication breakdowns. For some, the referral process comprised a series of barriers to be overcome, and there were additional hurdles for remote patients referred initially to local (non-specialist) hospitals. Rural patients appeared to be less demanding than their urban counterparts when evaluating their care, and this was extended to the acceptance of some clear transport problems. CONCLUSIONS: For patients, the most important components of cancer care were similar, whether rural or urban residents. The main differences for rural patients were lower expectations of care and more hurdles before reaching specialist care. These differences might explain the trend to more advanced disease at diagnosis in rural patients if they lead to significant delays. GPs have major influences on this and all the patients' priorities.

Can audit of a local protocol for the management of lipid disorders effect and detect a change in clinical practice?

OBJECTIVE: To audit the implementation of a local protocol for the management of lipid disorders in Grampian. INTERVENTION: A local protocol for the detection and management of lipid disorders, developed by a multi-disciplinary group and based on available national guidelines and systematic reviews, was disseminated and implemented using the audit cycle. DESIGN: An uncontrolled before and after study using four surveys of different aspects of lipid management in primary and secondary care. RESULTS: There was evidence of change in clinical practice following protocol implementation. Improvements in patient care were identified. However, some areas of inappropriate practice remained unaltered. Changes in dietary therapy indicated an improvement in the appreciation of the importance of appropriate dietary advice. There was an increase in the proportion of patients who were appropriately screened and managed, and appropriately referred to the lipid clinic. The health status of patients being screened/treated following guidelines was poorer suggesting a reduction in screening of low risk patients and an increase in lipid assessment of patients with other health problems or risk factors i.e. more appropriate screening. However, after the protocol was disseminated, general practitioners screened less often for secondary causes of hyperlipidaemia.

Developing a local protocol for the management of lipid disorders in general practice.

Guidelines and protocols are increasingly becoming part of routine clinical practice. A standardised methodology has been proposed for the development of national guidelines based on principles which are known to effect validity. However, less is known about the most appropriate methodology for the development of local protocols. A case study is presented of the development of a local protocol for the management of lipid disorders in primary care in Grampian. It highlights the practicalities of such a process and identifies lessons for others who may wish to develop local protocols.