National Diagnostic Laboratory Research Articles

The AlzMatch Pilot Study - Feasibility of Remote Blood Collection of Plasma Biomarkers for Preclinical Alzheimer’s Disease Trials

BackgroundAdvances in plasma biomarkers to detect Alzheimer’s disease (AD) biology allows researchers to improve the efficiency of participant recruitment into preclinical trials. Recently, protein levels of plasma amyloid-beta and tau proteins have been shown to be predictive of elevated amyloid in brain. Online registries, such as the Alzheimer’s Prevention Trials (APT) Webstudy, include and follow participants using remote assessments to facilitate efficient screening and enrollment of large numbers of individuals who may be at higher risk for AD.ObjectivesThe AlzMatch Pilot Study investigated the feasibility of recruiting individuals from an online registry for blood sample collection at community-based phlebotomy centers and plasma biomarker quantification to assess an individual’s eligibility for AD preclinical trials.DesignPilot feasibility study with co-primary outcomes.SettingThis pilot feasibility study included participants from the APT Webstudy, the remote assessment arm of the Trial-ready cohort for Preclinical and Prodromal AD (TRC-PAD) Platform. Novel design included collection of electronic consent, use of community laboratories for plasma collection, mass spectrometry-based biomarker assay, and telephone communication of plasma biomarker screening eligibility.ParticipantsParticipants invited to the AlzMatch pilot feasibility study were active in the APT Webstudy, 50 years of age or older, resided within 50 miles of both a Quest Diagnostics Patient Services Center (a national diagnostic laboratory with convenient locations for sample collection and processing) and one of six TRC-PAD vanguard clinical trial sites, had no self-reported dementia diagnosis, were able to communicate in English and engaged with the APT Webstudy within the prior 6 months.MeasurementsPrimary feasibility outcomes were completion of electronic consent (e-consent) for invited participants and collection of usable blood samples. Additional feasibility outcomes included invitation response rate, plasma biomarker eligibility status (based on amyloid beta-42/40 [Aβ42/40] concentration ratio), ApoE proteotype, and trial inclusion criterion), and completion of telephone contact to learn eligibility to screen for a study.Results300 APT Webstudy participants were invited to consent to the AlzMatch study. The AlzMatch e-consent rate was 39% (n=117) (95% CI of 33.5%–44.5%) overall, which was higher than the expected rate of 25%. Similar consent rates were observed across participants based on self-defined sex (41% Female (n=75), 37% Male (n=42)) and race and ethnicity (37% from underrepresented groups (URG) (n=36), 40% not from URG (n=79)). Among those that consented (n=117), plasma was successfully collected from 74% (n=87) (95% CI of 66%–82%), with similar rates across sex (76% Female (n=57), 71% Male (n=30)) and race and ethnicity (75% URG (n=27) and 75% not from URG (n=59)). 60% (n=51) of participants with plasma biomarker results were eligible to screen for future preclinical AD trials.ConclusionElectronic consent of participants through an online registry, blood sample collection at community-based centers, plasma biomarker quantification and reporting, and biomarker assessments for study eligibility were all feasible with similar engagement rates across demographic groups. Although this pilot was a small and selective sample, participants engaged and consented at higher than expected rates. We conclude that collecting blood at community laboratories for biomarker analyses may improve accessibility beyond research, and may facilitate broader access for clinical use of AD plasma biomarkers. Based on our results, an expanded version of the AlzMatch study is underway, which involves expanding invitations to additional APT Webstudy participants and clinical trial sites.

Assessing the use of diagnostic laboratory accession data to support national bovine viral diarrhoea control in New Zealand

ABSTRACTAims: To assess the suitability of using existing national diagnostic laboratory testing data to support national bovine viral diarrhoea (BVD) research, surveillance, and control in New Zealand.Methods: Data on laboratory accessions for BVD diagnostic testing in New Zealand from 1 January 2015 to 31 December 2017 were provided by four commercial veterinary diagnostic companies. The data were integrated into a single dataset containing the unique accession number, sample submission date, farm location (territorial authority level), test type (bulk milk antibody-ELISA, bulk milk PCR, serum antibody-ELISA, blood/serum/tissue antigen-ELISA, or blood/serum/tissue PCR), and test results. Estimates for the number of registered cattle farms in each territorial authority were generated from the National Animal Identification and Tracing database. Results were summarised for July 2015 to June 2016 and July 2016 to June 2017.Results: There was a total of 59,007 unique BVD diagnostic test accessions including 39,920 (67.6%) for bulk milk antibody-ELISA, 27,832 (47.2%) for bulk milk PCR, 3,229 (5.5%) for serum antibody-ELISA, 9,132 (15.5%) for blood/serum/tissue antigen-ELISA, and 7,122 (12.1%) for blood/serum/tissue PCR. Of the 17,946 accessions for blood/serum/tissue samples, 4,316 (24.0%) were missing the herd production type and 6,678 (37.2%) were missing the animals age. Approximately 7,000/10,958 (65%) dairy herds and 1,600/43,611 (4%) beef herds were conducting annual BVD screening tests. In 2016/2017, the prevalence of accessions with ≥1 BVD-positive result was 40.6% for bulk milk antibody, 6.4% for bulk milk PCR, 45.6% for serum antibody, and 9.8% for blood/serum/tissue antigen-ELISA or PCR tests. There was substantial regional variation in both the percentage of herds testing for BVD and the prevalence of positive accessions. Following pooled serum antibody-ELISA, only 175/604 (29.0%) beef herds and 177/566 (31.3%) dairy herds had recorded follow-up testing.Conclusions and Clinical Relevance: Laboratory diagnostic accession data has the potential to provide valuable insights about BVD epidemiology in New Zealand, but there are significant limitations in the data collected and discrepancies in the different systems that each laboratory uses to measure, interpret, and record diagnostic data. There is a strong need to develop a more consistent national system for recording and sharing BVD test results to support BVD management at farm and industry levels.Abbreviations: BVD: Bovine viral diarrhoea; Ct: Cycle threshold; NAIT: National Animal Identification and Tracing; NZVP: New Zealand Veterinary Pathology; PI: Persistently infected; S/P: Sample to positive control

034 Surgical wound healing in patients with epidermolysis bullosa

Despite limited evidence to suggest patients with Epidermolysis Bullosa (EB) have more postoperative wound complications than the general population, we have noted reluctance among some surgeons to operate on these patients. Patients recruited from the Australian National Diagnostic Laboratory Database for EB and the Australasian EB Registry were posted the ‘Surgical Wound and Scar Healing in EB’ questionnaire which contains twenty-two questions about a patients experience of having surgery. Forty-six patients reported a total of 94 surgical procedures. The most frequent surgical procedure was the excision of a skin lesion, followed by musculoskeletal surgery, a caesarean section or an appendectomy. Five patients reported blistering at the surgical wound site after seven surgeries. Four patients reported four postoperative wound infections and one reported a postoperative wound dehiscence. Twenty patients had 34 postoperative wounds which they felt healed slower than someone without EB and 30 patients had 55 postoperative wounds which they felt healed at the same rate as someone without EB. After 67 surgeries, the postoperative scar was reported to heal flat and after 18 it was reported to heal as a keloid scar. It is unlikely that patients with localised EB simplex will develop blistering at the postoperative wound site, but about a quarter of patients with generalised EB may develop blistering which is likely to interfere with wound healing. Postoperative wound infections do not appear to occur any more frequently in patients with EB than in patients within the general population and wound dehiscence is uncommon in patients with EB. Postoperative wounds may heal at a similar or slower rate in patients with EB compared to those within the general population and patients with EB may have a propensity to develop keloid scars. Despite the inherent limitations of a postal survey we feel clinicians should be more confident to refer patients with EB for surgery and surgeons reassured about postoperative wound healing and complications.

A Review of 52 Pedigrees with Epidermolysis Bullosa Simplex Identifying Ten Novel Mutations in KRT5 and KRT14 in Australia

Epidermolysis bullosa simplex (EBS) is a rare heritable skin fragility disorder, most commonly caused by dominant mutations in KRT5 and KRT14. EBS shows clinical heterogeneity with localised, intermediate and generalised severe forms, which tend to correlate with the location and nature of the disease causing mutations. We therefore aimed to identify the KRT5 and KRT14 mutations in patients diagnosed with EBS in Australia, and explore in depth the genotype to the phenotype correlations in patients with novel variants. Australian patients who were diagnosed with EBS after referral to the Australian National Diagnostic Laboratory for EB were offered mutation screening in the KRT5 and KRT14 genes. From this, 32 different mutations in KRT5 and KRT14 were identified within 39 of 52 pedigrees. Ten of these mutations from 9 different pedigrees were novel, a further fatal case caused by KRT5 E477K is reported and in addition the third reported case of digenic inheritance in EBS was also observed.

The distribution of epidermolysis bullosa in Australia with a focus on rural and remote areas.

Patients with epidermolysis bullosa (EB) require specialised medical care. In Australia this expertise is located in the major cities, with patients living in rural and remote areas having reduced access to these services. We aim to analyse the geographical distribution of patients with EB in Australia to determine the relevance of this potential geographical disadvantage for this population. Using postal codes obtained from the Australian National Diagnostic Laboratory Database for EB and the Australasian EB Registry, living patients with EB in Australia were categorised using the Australian standard geographical classification, remoteness areas. An analysis of EB subtype, including severity was also performed. A total of 318 patients were categorised, of whom 221 lived in major cities, 65 in inner regional areas, 26 in outer regional areas, four in remote and two in very remote areas. Half the patients living in remote and very remote areas had severe forms of EB. A significant proportion of patients with EB live outside the major cities in Australia. Half of the patients living in remote and very remote areas had severe forms of EB. Targeted strategies to improve access to EB-specific medical care may be needed for patients living in rural and remote areas.

Bacteria isolations from broiler and layer chicks in zambia.

Chick mortality (CM) is one of the major constraints to the expansion of the poultry industry in Zambia. Of the 2,829 avian disease cases submitted to the national diagnostic laboratory based at the Central Veterinary Research Institute in Lusaka between 1995 and 2007, 34.39% (973/2,829) were from CM cases. The disease accounted for 40.2% (218,787/544,903) mortality in the affected flocks with 89.6% (196,112/218,787) of the affected birds dying within seven days. Major bacteria species involved were Escherichia coli, Salmonella gallinarum, and Proteus species being isolated from 84.58%, 46.15%, and 26.93% of the reported CM cases (n = 973), respectively. Detection of Salmonella typhimurium, Salmonella enteritidis, and Salmonella dublin indicates that poultry has the potential of transmitting zoonotic pathogenic bacteria to humans. The proportion of Salmonella gallinarum reactors in the adult breeding stock was generally low (<0.5%) throughout the study period although its prevalence in CM cases was correlated (r = 0.68, P < 0.011) with seroprevalence of the same pathogen in the adult breeding stock. Given that the disease accounts for a large proportion of the avian diseases in Zambia as shown in the present study (34.39%, n = 2,829), it is imperative that an effective disease control strategy aimed at reducing its occurrence should be developed.

The current challenges of dourine: difficulties in differentiating Trypanosoma equiperdum within the subgenus Trypanozoon

During its 20th annual meeting in Paris in May 1999, the OIE (World organisation for animal health) Ad Hoc Group on Non-Tsetse Transmitted Animal Trypanosomoses expressed the following concerns about dourine: the discrepancies in some of the results of the complement fixation test (CFT), which is the only international diagnostic test officially recognised by the International Organisation for the Transportation of Equidae; the persistence of suspected cases of dourine in some Asian, European and African countries; the impossibility of differentiating Trypanosoma equiperdum from Trypanosoma evansi and of isolating new strains of T. equiperdum from clinical cases that have appeared in various parts of the world since 1982. In the light of these concerns, it was decided, in agreement with the Directorate of the Federal Veterinary Services of Russia in Moscow, to perform comparative trials on the value of CFT/dourine at the OIE Reference Laboratory for dourine in Moscow (The All-Russian Research Institute of Experimental Veterinary Medicine) using reagents (antigens and sera) from seven countries with extensive experience in the field of dourine diagnosis, namely, South Africa, France, Italy, Germany, Russia, the United States of America and the People's Republic of China. It is thanks to the successful co-operation of these countries that the trials were made possible. Results showed an overall concordance and were submitted for consideration to the OIE Biological Standards Commission, the commission which is in charge of the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. These trials serve as a starting point for further study, particularly in the following areas: the isolation of new strains of T. equiperdum from clinical dourine cases; the identification of specific markers for T. equiperdum which would make it possible to differentiate it from among the other species within the subgenus Trypanozoon; the experimental infection of horses with newly isolated T. equiperdum strains to compare their pathogenicity with those currently used in national diagnostic laboratories and with that of T. evansi; phylogenetic studies; the proposal and validation of new, internationally recognised diagnostic test(s) for dourine.

Central or national veterinary diagnostic laboratories.

Information on laboratory organisational structure and roles was gathered from twenty-four national veterinary diagnostic laboratories around the world. This information was used to develop an overview of the organisation and roles of the national veterinary diagnostic laboratory. In addition, the national diagnostic laboratory for the United States of America (the National Veterinary Services Laboratories) was used as an example to provide more information on the activities of such a laboratory. The survey revealed great variation in organisation and structure among the twenty-four laboratories. A few have only one facility, others function at multiple locations, and all have different supervisory reporting systems. However, these laboratories all form a significant part of the national veterinary infrastructure. All of the laboratories contacted in the survey report to (or are at least partially under the direction of) the Chief Veterinary Officer or another senior animal health official, and most report to the Ministry of Agriculture. The national veterinary diagnostic laboratories have similar roles, and are all responsible for foreign animal disease diagnosis and for providing support to national disease eradication or control programmes. Most also play an active role in import and export testing, either performing the tests themselves or developing a quality assurance programme for other laboratories that perform the testing. In addition, the national veterinary diagnostic laboratories usually undertake research and provide training, consultation, disease surveillance information and, in some cases, vaccine evaluation.

Update on the profile of multiple endocrine neoplasia type 2aRET mutations

BACKGROUND The RET protooncogene is responsible for the inherited cancer syndromes multiple endocrine neoplasia type 2a (MEN 2a) and familial medullary thyroid carcinoma (FMTC). Identification of predisposing germline RET mutations has allowed a DNA-based approach to diagnosis. Despite known mutational occurrences in RET exons 10,11,13, and 14, the majority of national diagnostic laboratories, with rare exception, continue to limit genetic analysis for MEN 2a and FMTC to RET exons 10 and 11. This study was undertaken to examine the genotype pattern of germline MEN 2a/FMTC RET mutations in an ethnically diverse North American population and assess the sensitivity of a more comprehensive testing approach to mutation detection that encompassed RET exons 10, 11, 13, and 14. METHODS The study group was comprised of 414 unselected consecutive patients at risk for hereditary MTC referred to a single laboratory. Genetic analysis used genomic DNA and a polymerase chain reaction-based denaturing gradient gel electrophoresis strategy for mutation detection in exons 10, 11, 13, and 14. RESULTS Thirty-five distinct MEN 2a or FMTC kindreds were identified. Thirteen different missense germline mutations were detected in 29 of the 35 families at RET codons 609, 618, 620, and 634 within exons 10 (31.4%) and 11 (51.4%). Of the 18 families with an exon 11 mutation, 12 (66.7%) harbored the most common TGCCGC missense change at codon 634. Four families (11.4%) harbored a codon 804 mutation in exon 14. Two of the codon 804 mutations uncovered were novel (GTGATG and GTGCTG) and occurred in three of the four families. Germline mutations were not observed in exon 13. A RET sequence abnormality remained undetected in two families with established multigenerational MTC. Hirschsprung's disease cosegregated in five families with MEN 2a. CONCLUSIONS The sensitivity of genetic screening for MEN 2a offered by diagnostic laboratories that limit RET analysis to exons 10 and 11 is 82.8%. Genetic testing that includes RET exon 14 results in a more complete and accurate analysis with a sensitivity approaching 95%. The authors recommend that clinicians confirm the comprehensiveness of a laboratory's genetic screening approach for MEN 2a to ensure thoroughness of sample analysis. Cancer 1997; 80:557-68. © 1997 American Cancer Society

Vibrio mimicus diarrhea following ingestion of raw turtle eggs.

Clinical and epidemiological characteristics of diarrhea associated with Vibrio mimicus were identified in 33 hospitalized patients referred to the Costa Rican National Diagnostic Laboratory Network between 1991 and 1994. The relevant symptoms presented by patients included abundant watery diarrhea, vomiting, and severe dehydration that required intravenous Dhaka solution in 83% of patients but not fever. Seroconversion against V. mimicus was demonstrated in four patients, from whom acute- and convalescent-phase sera were obtained. Those sera did not show cross-reaction when tested against Vibrio cholerae O1 strain VC-12. All the V. mimicus isolates from these cases produced cholera toxin (CT) and were susceptible to commonly used antibiotics. Attempts to isolate this bacterium from stool samples of 127 healthy persons were not successful. Consumption of raw turtle eggs was recalled by 11 of the 19 (58%) individuals interviewed. All but two V. mimicus diarrheal cases were sporadic. These two had a history of a common source of turtle (Lepidochelys olivacea) eggs for consumption, and V. mimicus was isolated from eggs from the same source (a local market). Among the strains, variations in the antimicrobial susceptibility pattern were observed. None of the strains recovered from market turtle eggs nor the four isolates from river water showed CT production. Further efforts to demonstrate the presence of CT-producing V. mimicus strains in turtle eggs were made. Successful results were obtained when nest eggs were tested. In this case, it was possible to isolate CT- and non-CT-producing strains, even from the same egg. For CT detection we used PCR, enzyme-linked immunosorbent assay (ELISA), and Y-1 cell assay, obtaining a 100% correlation between ELISA and PCR results. Primers Col-1 and Col-2, originally described as specific for the V. cholerae O1 ctxA gene, also amplified a 302-bp segment with an identical restriction map from V. mimicus. These results have important implications for epidemiological surveillance in tropical countries where turtle eggs are used for human consumption, serving as potential sources of cholera-like diarrhea.

Foot and mouth disease control strategies in Africa

Foot and mouth disease control strategies in Africa are complicated by special features of the disease and its control which are peculiar to Africa. Six of the seven serotypes of foot and mouth disease virus are known to occur in Africa and within each serotype wide antigenic variations have been demonstrated. This among other factors has necessitated constant changes in vaccine viruses. Recent serological studies indicated the possibility of rationalising the selection of vaccine viruses to a few baseline strains. Detailed epidemiological studies are lacking in view of the limited laboratory facilities. The necessity for a network of national diagnostic laboratories coordinated through an international regional FMD reference centre in Africa is stressed. The role of animal movements of different forms and wildlife mammals in virus dissemination is discussed. Examples of effective FMD control programmes in Northe, Eastern and Southern Africa are described. It is stressed that a greater effort will be required in regional coordination of FMD control programmes.