Conservation of vCJD Strain Properties After Extraction and In Vitro Propagation of PrPSc from Archived Formalin-Fixed Brain and Appendix Tissues Using Highly Sensitive Protein Misfolding Cyclic Amplification.

Three retrospective lymphoreticular tissue studies (Appendix I, II, and III) aimed to estimate the UK prevalence of variant Creutzfeldt-Jakob disease (vCJD), following exposure of the population to the bovine spongiform encephalopathy (BSE) agent, in the late 1980s and 1990s. These studies evaluated the presence of abnormal prion protein aggregates, in archived formalin-fixed paraffin-embedded (FFPE) appendectomy samples, by immunohistochemical detection. Although there was concordance in the estimated prevalence of vCJD from these studies, the identification of positive specimens from pre- and post-BSE-exposure periods in Appendix III study has raised questions regarding the nature and origin of the detected abnormal prion protein. We applied a robust and novel approach in the extraction of disease-associated prion protein (PrPSc) present in frozen and FFPE samples of brain and appendix from a patient with pathologically confirmed vCJD. The extracted material was used to seed the highly sensitive protein misfolding cyclic amplification assay (hsPMCA) to investigate the in vitro and in vivo propagation properties of the extracted abnormal prion protein. We demonstrate that PrPSc can be successfully extracted from FFPE appendix tissue and propagated in vitro. Bioassay in wild-type and gene-targeted mouse models confirmed that the extracted and amplified product is infectious and retains strain properties consistent with vCJD. This provides a highly sensitive and reliable platform for subsequent analysis of the archived FFPE appendix tissue derived from the Appendix II and III surveys, to further evaluate the nature of the abnormal PrP detected in the positive samples.

BSE can propagate in sheep co-infected or pre-infected with scrapie.

To understand the possible role of mixed-prion infections in disease presentation, the current study reports the co-infection of sheep with bovine spongiform encephalopathy (BSE) and scrapie. The bovine BSE agent was inoculated subcutaneously into sheep with ARQ/ARQ or VRQ/ARQ PRNP genotypes either at the same time as subcutaneous challenge with scrapie, or three months later. In addition, VRQ/VRQ sheep naturally infected with scrapie after being born into a scrapie-affected flock were challenged subcutaneously with BSE at eight or twenty one months-of-age. Sheep were analysed by incubation period/attack rate, and western blot of brain tissue determined the presence of BSE or scrapie-like PrPSc. Serial protein misfolding cyclic amplification (sPMCA) that can detect very low levels of BSE in the presence of an excess of scrapie agent was also applied to brain and lymphoreticular tissue. For VRQ/ARQ sheep challenged with mixed infections, scrapie-like incubation periods were produced, and no BSE agent was detected. However, whilst ARQ/ARQ sheep developed disease with BSE-like incubation periods, some animals had a dominant scrapie western blot phenotype in brain, but BSE was detected in these sheep by sPMCA. In addition, VRQ/VRQ animals challenged with BSE after natural exposure to scrapie had scrapie-like incubation periods and dominant scrapie PrPSc in brain, but one sheep had BSE detectable by sPMCA in the brain. Overall, the study demonstrates for the first time that for scrapie/BSE mixed infections, VRQ/ARQ sheep with experimental scrapie did not propagate BSE but VRQ/VRQ sheep with natural scrapie could propagate low levels of BSE, and whilst BSE readily propagated in ARQ/ARQ sheep it was not always the dominant PrPSc strain in brain tissue. Indeed, for several animals, a dominant scrapie biochemical phenotype in brain did not preclude the presence of BSE prion.

Risk Factors for Acute Rheumatic Fever: Literature Review and Protocol for a Case-Control Study in New Zealand.

Acute rheumatic fever (ARF) and its sequela, rheumatic heart disease (RHD), have largely disappeared from high-income countries. However, in New Zealand (NZ), rates remain unacceptably high in indigenous Maori and Pacific populations. The goal of this study is to identify potentially modifiable risk factors for ARF to support effective disease prevention policies and programmes. A case-control design is used. Cases are those meeting the standard NZ case-definition for ARF, recruited within four weeks of hospitalisation for a first episode of ARF, aged less than 20 years, and residing in the North Island of NZ. This study aims to recruit at least 120 cases and 360 controls matched by age, ethnicity, gender, deprivation, district, and time period. For data collection, a comprehensive pre-tested questionnaire focussed on exposures during the four weeks prior to illness or interview will be used. Linked data include previous hospitalisations, dental records, and school characteristics. Specimen collection includes a throat swab (Group A Streptococcus), a nasal swab (Staphylococcus aureus), blood (vitamin D, ferritin, DNA for genetic testing, immune-profiling), and head hair (nicotine). A major strength of this study is its comprehensive focus covering organism, host and environmental factors. Having closely matched controls enables the examination of a wide range of specific environmental risk factors.

The challenges and opportunities of translating best practice immunisation strategies among low performing general practices to reduce equity gaps in childhood immunisation coverage in New Zealand.

BACKGROUND: Immunisation coverage rates vary considerably at the local level across New Zealand and challenges remain with effectively translating best available research evidence into public health practice. This study aimed to translate best practices from high performing general practices into strategies to improve childhood immunisation coverage among low performing practices. METHODS: An intervention study was undertaken of general practices with low immunisation coverage rates and a high percentage of the enrolled population being of Maori ethnicity. Intervention groups received customised action plans and support for a 12 month period while control groups received 'business as usual' support. Structured interviews were conducted with key informants from all participating practices to understand current aspects related to childhood immunisation delivery and surveys were conducted to understand how the intervention worked. Collected data were thematically analysed. RESULTS: Ten sites were randomised to either intervention (n = 6) or control group (n = 4). Positive aspects of childhood immunisation delivery included high prioritisation at the practice and staff being pro-immunisation and knowledgeable. Key challenges experienced included inaccurate family contact information and discrepancies with referral processes to other providers. Other challenges noted were building rapport with families and vaccine hesitancy. The action plans included various strategies aimed to improve processes at the practice, contact and engagement with parents, and partnership development with local service providers. CONCLUSIONS: Creating customised action plans and providing support to providers were considered as helpful approaches when attempting to improve childhood immunisation coverage rates. Our study supports the notion that one strategy will not solely by itself improve childhood immunisation rates and highlights the importance of having a toolkit of strategies from which to draw from.

Identifying factors behind the general practice use of the term 'decline' for the childhood immunisation programme in New Zealand.

INTRODUCTION The role of healthcare providers and their use of systems is one of the most important factors in vaccination uptake. AIM To identify reasons and find patterns behind why immunisation providers code the word 'decline' in their system for childhood immunisation events. METHODS A qualitative study involving face-to-face semi-structured interviews with staff members involved in immunisation delivery. General practices were purposively selected for having either high or low rates of registered children coded on the electronic practice management system as having declined immunisation events. Thematic analysis was undertaken using an inductive approach to link themes to the data. RESULTS A total of 35 interviews were conducted with practice nurses; 21 were from practices with high rates of registered children recorded as having declining immunisation events, and 14 practices with low rates of declining. Common themes were: effective use of systems, early and ongoing engagement, adequate health care practitioner time and practitioner experience. Practices with low rates of coded decliners had stronger approaches for early and ongoing engagement, and were less likely to use formalised decline forms. As practice immunisation coverage rates improved over time, there was perceived to be less expressed vaccine hesitancy from families. CONCLUSIONS The reasons for coding individuals as 'decliners' are a complex mixture of individual, community, practitioner and practice systems. Front-line providers need adequate tools, time and resourcing to support effective and ongoing engagement with families. Community factors have influence but can change over time.

Stability of murine scrapie strain 87V after passage in sheep and comparison with the CH1641 ovine strain.

Breed- and prion protein (PRNP) genotype-related disease phenotype variability has been observed in sheep infected with the 87V murine scrapie strain. Therefore, the stability of this strain was tested by inoculating sheep-derived 87V brain material back into VM mice. As some sheep-adapted 87V disease phenotypes were reminiscent of CH1641 scrapie, transgenic mice (Tg338) expressing ovine prion protein (PrP) were inoculated with the same sheep-derived 87V sources and with CH1641. Although at first passage in VM mice the sheep-derived 87V sources showed some divergence from the murine 87V control, all the characteristics of murine 87V infection were recovered at second passage from all sheep sources. These included 100 % attack rates and indistinguishable survival times, lesion profiles, immunohistochemical features of disease-associated PrP accumulation in the brain and PrP biochemical properties. All sheep-derived 87V sources, as well as CH1641, were transmitted to Tg338 mice with identical clinical, pathological, immunohistochemical and biochemical features. While this might potentially indicate that sheep-adapted 87V and CH1641 are the same strain, profound divergences were evident, as murine 87V was unable to infect Tg338 mice but was lethal for VM mice, while the reverse was true for CH1641. These combined data suggest that: (i) murine 87V is stable and retains its properties after passage in sheep; (ii) it can be isolated from sheep showing a CH1641-like or a more conventional scrapie phenotype; and (iii) sheep-adapted 87V scrapie, with conventional or CH1641-like phenotype, is biologically distinct from experimental CH1641 scrapie, despite the fact that they behave identically in a single transgenic mouse line.

Archival search for historical atypical scrapie in sheep reveals evidence for mixed infections.

Natural scrapie in sheep occurs in classical and atypical forms, which may be distinguished on the basis of the associated neuropathology and properties of the disease-associated prion protein on Western blots. First detected in 1998, atypical scrapie is known to have occurred in UK sheep since the 1980s. However, its aetiology remains unclear and it is often considered as a sporadic, non-contagious disease unlike classical scrapie which is naturally transmissible. Although atypical scrapie tends to occur in sheep of prion protein (PRNP) genotypes that are different from those found predominantly in classical scrapie, there is some overlap so that there are genotypes in which both scrapie forms can occur. In this search for early atypical scrapie cases, we made use of an archive of fixed and frozen sheep samples, from both scrapie-affected and healthy animals (∼1850 individuals), dating back to the 1960s. Using a selection process based primarily on PRNP genotyping, but also on contemporaneous records of unusual clinical signs or pathology, candidate sheep samples were screened by Western blot, immunohistochemistry and strain-typing methods using tg338 mice. We identified, from early time points in the archive, three atypical scrapie cases, including one sheep which died in 1972 and two which showed evidence of mixed infection with classical scrapie. Cases with both forms of scrapie in the same animal as recognizable entities suggest that mixed infections have been around for a long time and may potentially contribute to the variety of scrapie strains.

Characteristics and behaviour of screen-detected ductal carcinoma in situ of the breast: comparison with symptomatic patients.

Breast cancer is the most common malignancy in Singapore women. Ductal carcinoma in situ (DCIS) is the putative, non-obligate precursor of the majority of invasive breast cancers. The efficacy of the Singapore breast-screening pilot project in detecting early stage breast cancer led to the launch of a national breast-screening programme, BreastScreen Singapore (BSS), in January 2002. In this study, we compared clinicopathological and immunohistochemical characteristics, as well as clinical outcomes, between screen-detected and symptomatic DCIS. The study cohort comprised 1202 cases of DCIS diagnosed at Singapore General Hospital from 1994 to 2010. Comparison of clinicopathological parameters, immunohistochemical results of ER, PR, HER2, CK14, EGFR, and 34ßE12, and clinical outcomes was carried out between the 2 groups. Amongst 1202 cases, 610 (50.7%) were screen-detected and 592 (49.3%) were symptomatic DCIS. Screen-detected cases were smaller in size (P < 0.001), of lower nuclear grade (P = 0.004), and more frequently expressed ER (P < 0.001). Luminal A phenotype was more frequently observed in screen-detected DCIS, while triple-negative and HER2 phenotypes were more common in symptomatic DCIS (P < 0.001). The basal-like phenotype was also more frequent in symptomatic DCIS (P = 0.041). Mean and median follow-up was 99.7 and 97.8 months, respectively, with a maximum follow-up of 246.0 months. More symptomatic patients developed invasive recurrences compared to screen-detected patients (P = 0.001). A trend for better disease-free survival was observed in screen-detected patients (P = 0.076). Patients who were screen-detected experienced better overall survival than those with symptomatic DCIS (P = 0.007). Our data indicate a more favourable outcome of screen-detected DCIS patients confirming the role of BSS in early identification of this curable disease.

MED12 is frequently mutated in breast phyllodes tumours: a study of 112 cases.

AIM: To determine the frequency of MED12 mutations in a series of 112 breast phyllodes tumours, and to correlate the findings with clinicopathological parameters and survival outcomes. METHODS: Phyllodes tumours from the Department of Pathology, Singapore General Hospital, were classified into benign, borderline and malignant categories. Genomic DNA from formalin-fixed paraffin-embedded phyllodes tumours was extracted, purified and subjected to ultra-deep-targeted amplicon sequencing across exon 2 of the MED12 gene. Sequencing was performed on the Illumina MiSeq next-generation sequencing platform and bioinformatics analysis applied. Appropriate statistical analyses were carried out. RESULTS: There were 66 benign, 32 borderline and 14 malignant tumours, with 43 (65.1%), 21 (65.6%) and 6 (42.8%) disclosing MED12 mutations (missense, splice site, indel), respectively. For 97 cases with available follow-up, there were 10 (10.3%) recurrences. Patients with phyllodes tumours that harboured MED12 mutations experienced improved disease-free survivals, with higher recurrence likelihood in those without MED12 mutations (HR 9.99, 95% CIs 1.55 to 64.42, p=0.015). CONCLUSIONS: Similar to fibroadenomas, phyllodes tumours show a high frequency of MED12 mutations, affirming the close biological relationship between these fibroepithelial neoplasms.

eCHAT for lifestyle and mental health screening in primary care.

PURPOSE: Early detection and management of unhealthy behaviors and mental health issues in primary care has the potential to prevent or ameliorate many chronic diseases and increase patients' well-being. This study aimed to assess the feasibility and acceptability of the systematic use of a Web-based eCHAT (electronic Case-finding and Help Assessment Tool) screening patients for problematic drinking, smoking, and other drug use, gambling, exposure to abuse, anxiety, depression, anger control, and physical inactivity, and whether they want help with these issues. Patients self-administered eCHAT on an iPad in the waiting room and received summarized results, including relevant scores and interpretations, which could be by a family physician on the website and in the electronic health record (EHR) at the point of care. METHODS: We conducted a mixed method feasibility and acceptability study in 2 general practices in Auckland, New Zealand. Participants were consecutive adult patients attending the practice during a 2-week period, as well as all practice staff. Patients completed eCHAT, doctors accessed the summarized reports. Outcome measures were patients' responses to eCHAT, and patients' written and staff recorded interview feedback. RESULTS: Of the 233 invited patients, 196 (84%) completed eCHAT and received feedback. Domains where patients wanted immediate help were anxiety (9%), depression (7%), physical activity (6%), and smoking (5%), which was not overwhelming for physicians to address. Most patients found the iPad easy to use, and the questions easy to understand and appropriate; they did not object to questions. Feedback from 7 doctors, 2 practice managers, 4 nurses, and 5 receptionists was generally positive. Practices continue to use eCHAT regularly since the research was completed. CONCLUSIONS: eCHAT is an acceptable and feasible means of systemic screening patients for unhealthy behaviors and negative mood states and is easily integrated into the primary care electronic health record.

When is acute persistent cough in school-age children and adults whooping cough? A prospective case series study.

BACKGROUND: Pertussis is a vaccine modified disease in most age groups and hence subtle in its presentation. Current diagnostic approaches require relatively invasive sampling. AIM: To determine the incidence of B. pertussis infection among people aged 5-49 years identified in primary care with acute persistent cough using an oral fluid based diagnostic test. DESIGN AND SETTING: Active surveillance of acute persistent cough of 2 weeks duration or greater was established in Auckland, New Zealand from May to October 2011. The 15 participating primary care practices provided care for a socioeconomically diverse population. METHOD: Recent B. pertussis infection was determined by measurement of IgG antibodies to pertussis toxin (PT) in an oral fluid sample. An IgG antibody titre to PT of ≥70 arbitrary units defined recent infection. Participants reported symptoms at presentation and kept a cough diary. RESULTS: A total of 226 participants were enrolled: 70 (31%) were children (5-16 years) and 156 (69%) were adults (17-49 years). Oral fluid samples were obtained from 225 participants. Ten per cent (23/225) had recent B. pertussis infection including a larger proportion of children than adults (17% versus 7%, P = 0.003). Neither cough duration nor any individual symptom discriminated between those with and without recent B. pertussis infection. CONCLUSION: Pertussis is a frequent cause of acute persistent cough presenting to primary care. Clinical differentiation of pertussis from other causes of acute persistent cough is difficult. An oral fluid based diagnostic test, which is less invasive than other diagnostic approaches, has high acceptability in primary care.

Susceptibility of young sheep to oral infection with bovine spongiform encephalopathy decreases significantly after weaning.

Bovine spongiform encephalopathy (BSE) is a transmissible spongiform encephalopathy (TSE) (or prion disease) that is readily transmissible to sheep by experimental infection and has the shortest incubation period in animals with the ARQ/ARQ PRNP genotype (at codons 136, 154, and 171). Because it is possible that sheep in the United Kingdom could have been infected with BSE by being fed contaminated meat and bone meal supplements at the same time as cattle, there is considerable interest in the responses of sheep to BSE inoculation. Epidemiological evidence suggests that very young individuals are more susceptible to TSE infection; however, this has never been properly tested in sheep. In the present study, low doses of BSE were fed to lambs of a range of ages (~24 h, 2 to 3 weeks, 3 months, and 6 months) and adult sheep. The incidence of clinical BSE disease after inoculation was high in unweaned lambs (~24 h and 2 to 3 weeks old) but much lower in older weaned animals The incubation period was also found to be influenced by the genotype at codon 141 of the PRNP gene, as lambs that were LF heterozygotes had a longer mean incubation period than those that were homozygotes of either type. The results suggest that sheep in the United Kingdom would have been at high risk of BSE infection only if neonatal animals had inadvertently ingested contaminated supplementary foodstuffs.

False-negative bone scan and choline pet/ct study in a case of prostate cancer: the pitfall of the small cell prostate carcinoma variant.

We present a rare variant of prostate carcinoma. The patient is a 45-year-old male with elevated prostate-specific antigen levels at screening. Magnetic resonance imaging revealed hyperenhancing lesions throughout the axial skeleton. The fluorine-18 fluorocholine (FCH) positron emission tomography/computed tomography (PET/CT) scan showed no abnormal bone findings. Subsequently, a technetium-99 methydiphosphonate (Tc99m-MDP) bone scan was performed, with additional correlative single-photon emission computed tomography (SPECT)/CT imaging of the pelvis and the results were essentially normal. A percutaneous core biopsy of one of the bone lesions in L5 was performed and histology confirmed small cell (neuroendocrine) variant of prostate cancer. Our case illustrates a possible pitfall in molecular imaging of prostate carcinomas, whereby both bone scintigraphy and FCH PET/CT scans showed no definite bone lesions to correlate with marrow signal abnormalities seen on MR imaging. This highlights the need for caution in the diagnostic evaluation of prostate cancers with known small cell variants.

Discovery of amide replacements that improve activity and metabolic stability of a bis-amide smoothened antagonist hit.

A bis-amide antagonist of Smoothened, a seven-transmembrane receptor in the Hedgehog signaling pathway, was discovered via high throughput screening. In vitro and in vivo experiments demonstrated that the bis-amide was susceptible to N-acyl transferase mediated amide scission. Several bioisosteric replacements of the labile amide that maintained in vitro potency were identified and shown to be metabolically stable in vitro and in vivo.

Increased susceptibility of human-PrP transgenic mice to bovine spongiform encephalopathy infection following passage in sheep.

The risk of the transmission of ruminant transmissible spongiform encephalopathy (TSE) to humans was thought to be low due to the lack of association between sheep scrapie and the incidence of human TSE. However, a single TSE agent strain has been shown to cause both bovine spongiform encephalopathy (BSE) and human vCJD, indicating that some ruminant TSEs are transmissible to humans. While the transmission of cattle BSE to humans in transgenic mouse models has been inefficient, indicating the presence of a significant transmission barrier between cattle and humans, BSE has been transmitted to a number of other species. Here, we aimed to further investigate the human transmission barrier following the passage of BSE in a sheep. Following inoculation with cattle BSE, gene-targeted transgenic mice expressing human PrP showed no clinical or pathological signs of TSE disease. However, following inoculation with an isolate of BSE that had been passaged through a sheep, TSE-associated vacuolation and proteinase K-resistant PrP deposition were observed in mice homozygous for the codon 129-methionine PRNP gene. This observation may be due to higher titers of the BSE agent in sheep or an increased susceptibility of humans to BSE prions following passage through a sheep. However, these data confirm that, contrary to previous predictions, it is possible that a sheep prion is transmissible to humans and that BSE from other species is a public health risk.

Addressing PXR liabilities of phthalazine-based hedgehog/smoothened antagonists using novel pyridopyridazines.

Pyridopyridazine antagonists of the hedgehog signaling pathway are described. Designed to optimize our previously described phthalazine smoothened antagonists, a representative compound eliminates a PXR liability while retaining potency and in vitro metabolic stability. Moreover, the compound has improved efficacy in a hedgehog/smoothened signaling mouse pharmacodynamic model.

Design of 1-piperazinyl-4-arylphthalazines as potent Smoothened antagonists.

The Hedgehog (Hh) signaling pathway regulates cell proliferation and differentiation in developing tissues, and abnormal activation of the Hh pathway has been linked to several tumor subsets. As a transducer of Hh signaling, the GPCR-like protein Smoothened (Smo) is a promising target for disruption of unregulated Hh signaling. A series of 1-amino-4-arylphthalazines was developed as potent and orally bioavailable inhibitors of Smo. A representative compound from this class demonstrated significant tumor volume reduction in a mouse medulloblastoma model.

Triple negative breast cancer: outcome correlation with immunohistochemical detection of basal markers.

We earlier evaluated the relationship of 653 triple negative breast cancers (TNBC) with basal immunophenotypic expression by using antibodies to basal cytokeratins (CK5/6, CK14, CK17, 34betaE12), p63, smooth muscle actin (SMA), epidermal growth factor receptor, and CD117, and found that a triple panel of CK14, 34betaE12 and epidermal growth factor receptor determined 84% of our cases to be basal-like. Women with basal-like TNBC tended to be younger (P=0.04), have histologically higher-grade tumors (P=0.047), with positive nodal status (P=0.047), than those whose tumors were nonbasal-like. Using univariate Cox regression analysis, tumor size (P=0.003), histologic grade (P=0.006), and nodal status (P=0.017) were significant factors for disease-free survival (DFS) among TNBC, whereas age (P=0.004), tumor size (P=0.001), histologic grade (P<0.001), nodal status (P=0.011), lymphovascular invasion (P=0.032), and pushing borders (P=0.042) were important for overall survival (OS). On multivariate analysis, age was statistically significant for both DFS and OS (P=0.033, 0.001 respectively), whereas histologic grade was important for OS (P<0.001). Kaplan Meier curves showed CK17 positivity to impact adversely on DFS (P=0.003) and OS (P=0.014), whereas CD117 positive staining was accompanied by diminished OS (P=0.036). SMA expression in TNBC however, revealed a trend for improved DFS (P=0.05). Our findings indicate that basal-like TNBC are associated with adverse clinicopathologic parameters, and that individual biologic markers of CK17, CD117, and SMA have prognostic implications on survival. Possibilities exist for future targeted therapy for this challenging group of breast cancers.