Stability during cooking of anthelmintic veterinary drug residues in beef

Anthelmintic drugs are widely used for treatment of parasitic worms in livestock, but little is known about the stability of their residues in food under conventional cooking conditions. As part of the European Commissionfunded research project ProSafeBeef, cattle were medicated with commercially available anthelmintic preparations, comprising 11 active ingredients (corresponding to 21 marker residues). Incurred meat and liver were cooked by roasting (40 min at 190°C) or shallow frying (muscle 8-12 min, liver 14-19 min) in a domestic kitchen. Raw and cooked tissues and expressed juices were analysed using a novel multi-residue dispersive solid-phase extraction method (QuEChERS) coupled with ultra-performance liquid chromatography-tandem mass spectrometry. After correction for sample weight changes during cooking, no major losses were observed for residues of oxyclozanide, clorsulon, closantel, ivermectin, albendazole, mebendazole or fenbendazole. However, significant losses were observed for nitroxynil (78% in fried muscle, 96% in roast muscle), levamisole (11% in fried muscle, 42% in fried liver), rafoxanide (17% in fried muscle, 18% in roast muscle) and triclabendazole (23% in fried liver, 47% in roast muscle). Migration of residues from muscle into expressed cooking juices varied between drugs, constituting 0% to 17% (levamisole) of total residues remaining after cooking. With the exception of nitroxynil, residues of anthelmintic drugs were generally resistant to degradation during roasting and shallow frying. Conventional cooking cannot, therefore, be considered a safeguard against ingestion of residues of anthelmintic veterinary drugs in beef. © 2011 Taylor & Francis.

Fasciola hepatica:Histological demonstration of apoptosis in the reproductive organs of flukes of triclabendazole-sensitive and triclabendazole-resistant isolates, and in field-derived flukes from triclabendazole-treated hosts, using in situ hybridisation to visualise endonuclease-generated DNA strand breaks

Investigation of the triclabendazole (TCBZ) resistance status of populations of Fasciola hepatica in field cases of fasciolosis, where treatment failure has been reported, can be supported by histological examination of flukes collected from recently treated hosts. In TCBZ-sensitive flukes (TCBZ-S) exposed to TCBZ metabolites for 1-4. days in vivo, but not in TCBZ-resistant flukes (TCBZ-R), morphological changes suggestive of apoptosis occur in cells undergoing meiosis or mitosis in the testis, ovary and vitelline follicles. In order to verify or refute the contention that efficacy of TCBZ treatment is associated with apoptosis in the reproductive organs of flukes, histological sections of TCBZ-S (Cullompton isolate) flukes and TCBZ-R (Sligo isolate) flukes were subjected to the TdT-mediated dUDP nick end labelling (TUNEL) in situ hybridisation method, a commercially available test specifically designed to label endonuclease-induced DNA strand breaks associated with apoptosis. Additionally, sections of in vivo-treated and untreated flukes originating from field outbreaks of suspected TCBZ-S and TCBZ-R fasciolosis were labelled by the TUNEL method. It was found that in treated TCBZ-S flukes, strong positive labelling indicating apoptosis was associated with morphologically abnormal cells undergoing mitosis or meiosis in the testis, ovary and vitelline follicles. Background labelling in the positive testis sections was attributed to heterophagy of cell debris by the sustentacular tissue. The triggering of apoptosis was probably related to failure of spindle formation at cell division, supporting the contention that TCBZ inhibits microtubule formation. In treated TCBZ-R (Sligo Type 1) flukes, and in treated flukes from field outbreaks of suspected TCBZ-R fasciolosis, no significant labelling was observed, while sections of fluke derived from a field case of fasciolosis where TCBZ resistance was not suspected were heavily labelled. Light labelling was associated with the testis of untreated Cullompton (TCBZ-S) and Sligo Type 2 (TCBZ-R) flukes, which exhibit abnormal spermatogenesis and spermiogenesis, respectively. This was attributed to apoptosis and to heterophagy of effete germ line cells by the sustentacular tissue. It is concluded that demonstration of apoptosis by in situ hybridisation using the TUNEL method on sections of 1-4. days in vivo TCBZ-treated F. hepatica can contribute to the diagnosis of TCBZ resistance in field outbreaks of fasciolosis. © 2012 Elsevier B.V.

Illness perception clusters at diagnosis predict psychological distress among women with breast cancer at 6 months post-diagnosis.

OBJECTIVE:

This study aimed to examine the extent to which illness perceptions and coping strategies among women diagnosed with breast cancer explain psychological distress at diagnosis and at 6?months post diagnosis relative to demographic and illness-related variables.

METHODS:

Women were recruited to the study shortly after diagnosis. A total of 90 women completed study materials (Illness Perception Questionnaire-Revised, the Cancer Coping Questionnaire and the Hospital Anxiety and Depression Scale) at time 1. The same questionnaires were sent approximately 6?months later to those who had consented at time 1, and completed questionnaires were returned by 72 women.

RESULTS:

Cluster analysis was used to identify groups of respondents who reported a similar profile of illness perception scores. Regression analysis demonstrated that one of these clusters was more likely to experience psychological distress than the other both at diagnosis and at 6?months post diagnosis. Illness perception cluster membership and positive focus type coping were the most important and consistent predictors of lower psychological distress at diagnosis and at 6?months post diagnosis.

CONCLUSIONS:

Illness perceptions remained relatively stable over the study period, and therefore we are unable to clarify whether changes in illness cognitions are associated with a corresponding change in psychological symptoms. Future research should evaluate the impact on psychological distress of interventions specifically designed to modify illness cognitions among women with breast cancer.

Investigation of the Human Brain Metabolome to Identify Potential Markers for Early Diagnosis and Therapeutic Targets of Alzheimer’s Disease

A study combining high resolution mass spectrometry (liquid chromatography-quadrupole time-of-flight-mass spectrometry, UPLC-QTof-MS) and chemometrics for the analysis of post-mortem brain tissue from subjects with Alzheimer’s disease (AD) (n = 15) and healthy age-matched controls (n = 15) was undertaken. The huge potential of this metabolomics approach for distinguishing AD cases is underlined by the correct prediction of disease status in 94–97% of cases. Predictive power was confirmed in a blind test set of 60 samples, reaching 100% diagnostic accuracy. The approach also indicated compounds significantly altered in concentration following the onset of human AD. Using orthogonal partial least-squares discriminant analysis (OPLS-DA), a multivariate model was created for both modes of acquisition explaining the maximum amount of variation between sample groups (Positive Mode-R2 = 97%; Q2 = 93%; root mean squared error of validation (RMSEV) = 13%; Negative Mode-R2 = 99%; Q2 = 92%; RMSEV = 15%). In brain extracts, 1264 and 1457 ions of interest were detected for the different modes of acquisition (positive and negative, respectively). Incorporation of gender into the model increased predictive accuracy and decreased RMSEV values. High resolution UPLC-QTof-MS has not previously been employed to biochemically profile post-mortem brain tissue, and the novel methods described and validated herein prove its potential for making new discoveries related to the etiology, pathophysiology, and treatment of degenerative brain disorders.

Availability of stage at diagnosis, cancer treatment delay and compliance with cancer guidelines as cancer registry indicators for cancer care in Europe:Results of EUROCHIP-3 survey

EUROCHIP (European Cancer Health Indicators Project) focuses on understanding inequalities in the cancer burden, care and survival by the indicators "stage at diagnosis," "cancer treatment delay" and "compliance with cancer guidelines" as the most important indicators. Our study aims at providing insight in whether cancer registries collect well-defined variables to determine these indicators in a comparative way. Eighty-six general European population-based cancer registries (PBCR) from 32 countries responded to the questionnaire, which was developed by EUROCHIP in collaboration with ENCR (European Network of Cancer Registries) and EUROCOURSE. Only 15% of all the PBCR in EU had all three indicators available. The indicator "stage at diagnosis" was gathered for at least one cancer site by 81% (using TNM in 39%). Variables for the indicator "cancer treatment delay" were collected by 37%. Availability of type of treatment (30%), surgery date (36%), starting date of radiotherapy (26%) and starting date of chemotherapy (23%) resulted in 15% of the PBCRs to be able to gather the indicator "compliance to guidelines". Lack of data source access and qualified staff were the major reasons for not collecting all the variables. In conclusion, based on self-reporting, a few of the participating PBCRs had data available which could be used for clinical audits, evaluation of cancer care projects, survival and for monitoring national cancer control strategies. Extra efforts should be made to improve this very efficient tool to compare cancer burden and the effects of the national cancer plans over Europe and to learn from each other. © 2012 UICC.

Molecular diagnosis of the myeloproliferative neoplasms: UK guidelines for the detection of JAK2 V617F and other relevant mutations

Molecular genetic assays for the detection of the JAK2 V617F (c.1849G>T) and other pathogenetic mutations within JAK2 exon 12 and MPL exon 10 are part of the routine diagnostic workup for patients presenting with erythrocytosis, thrombocytosis or otherwise suspected to have a myeloproliferative neoplasm. A wide choice of techniques are available for the detection of these mutations, leading to potential difficulties for clinical laboratories in deciding upon the most appropriate assay, which can lead to problems with inter-laboratory standardization. Here, we discuss the most important issues for a clinical diagnostic laboratory in choosing a technique, particularly for detection of the JAK2 V617F mutation at diagnosis. The JAK2 V617F detection assay should be both specific and sensitive enough to detect a mutant allele burden as low as 13%. Indeed, the use of sensitive assays increases the detection rate of the JAK2 V617F mutation within myeloproliferative neoplasms. Given their diagnostic relevance, it is also beneficial and relatively straightforward to screen JAK2 V617F negative patients for JAK2 exon 12 mutations (in the case of erythrocytosis) or MPL exon 10 mutations (thrombocytosis or myelofibrosis) using appropriate assays. Molecular results should be considered in the context of clinical findings and other haematological or laboratory results.

Guideline for the diagnosis and management of myelofibrosis

The guideline group regarding the diagnosis and management of myelofibrosis was selected to be representative of UK-based medical experts, together with a contribution from a single expert from the USA. MEDLINE and EMBASE were searched systematically for publications in English from 1966 until August 2011 using a variety of key words. The writing group produced the draft guideline, which was subsequently revised by consensus of the members of the General Haematology and Haemato-oncology Task Forces of the British Committee for Standards in Haematology (BCSH). The guideline was then reviewed by a sounding board of UK haematologists, the BCSH and the British Society for Haematology Committee and comments incorporated where appropriate. The criteria used to state levels and grades of evidence are as outlined in the Procedure for Guidelines commissioned by the BCSH; the 'GRADE' system was used to score strength and quality of evidence. The objective of this guideline is to provide healthcare professionals with clear guidance on the investigation and management of primary myelofibrosis, as well as post-polycythaemic myelofibrosis (post-PV MF) and post-thrombocythemic myelofibrosis (post-ET MF) in both adult and paediatric patients.

Fundus autofluorescence in the diagnosis of cystoid macular oedema

Aim: To determine the sensitivity and specificity of the non-invasive imaging technique, fundus autofluorescence (AF), in the diagnosis of cystoid macular oedema (CMO), using fluorescein angiography as the reference standard. Design: Retrospective, consecutive, observational case series. Methods: Ninety-six consecutive patients with CMO suspected clinically were selected from the AF database of the Retina Unit, Ophthalmology Department, Grampian University Hospitals-NHS Trust, between August 2004 and June 2006. Only patients in whom CMO was secondary to (1) cataract extraction, (2) inherited retinopathies, (3) inflammatory eye disease or (4) idiopathic cases were included in this study. Only patients in whom AF images had been performed within 2 weeks of FFA and, when obtained following FFA, there was a minimum gap of 4 days ("washing out" period), were considered eligible for this study. A total of 34 eyes from 34 patients were eligible and were included in this study. FFA was used as the reference test to confirm the presence of CMO, and, based on fluorescein angiography (FFA), CMO was graded as either mild or florid. AF images were examined in a masked fashion for the presence or absence of CMO. The sensitivity and specificity of AF in detecting CMO were then calculated. Results: CMO was seen on AF imaging as round or oval areas at the fovea with an AF signal similar to that of background levels. At this site (fovea), the AF signal is usually reduced compared with background, due to the blockage caused by luteal pigment. The diagnosis of CMO based on AF imaging had 81% sensitivity and 69% specificity when compared with the reference standard FFA. Based on the FFA, there were 12 cases of florid CMO and eight of mild CMO. Of the former, CMO was detected with AF imaging in 100% (12/12 eyes), and of the latter, in 50% (4/8 eyes). Conclusions: AF imaging can be used as a rapid, non-invasive technique in the diagnosis of CMO.

Diagnosis and management of congenital and idiopathic erythrocytosis

An erythrocytosis occurs when there is an increased red-cell mass. The causes of erythrocytosis are divided into primary, when there is an intrinsic defect in the erythroid cell, and secondary, when the cause is extrinsic to the erythroid cell. An idiopathic erythrocytosis occurs when the increased red-cell mass has no identifiable cause. Primary and secondary defects can be further classified as either congenital or acquired causes. The diagnostic pathway starts with a careful history and examination followed by measurement of the erythropoietin (EPO) levels. This allows a division of those patients with a low EPO level, who can then be investigated for primary causes of erythrocytosis, and those with a normal or high EPO level, where the oxygen-sensing pathway needs to be explored further. Physiological studies in those with congenital defects in the oxygen-sensing pathway show many changes in the downstream metabolism adapting to the defect, which has a bearing on the management of the disorders. Low-dose aspirin and venesection to an achievable target are the main therapeutic options that can be considered in the management of erythrocytosis. Specific guidance on venesection options should be considered with certain causes such as high oxygen-affinity hemoglobins.

PROSTATIC INTRAEPITHELIAL NEOPLASIA (PIN) - PERFORMANCE OF BAYESIAN BELIEF NETWORK FOR DIAGNOSIS AND GRADING

Prostatic intraepithelial neoplasia (PIN) diagnosis and grading are affected by uncertainties which arise from the fact that almost all knowledge of PIN histopathology is expressed in concepts, descriptive linguistic terms, and words. A Bayesian belief network (BBN) was therefore used to reduce the problem of uncertainty in diagnostic clue assessment, while still considering the dependences between elements in the reasoning sequence. A shallow network was used with an open-tree topology, with eight first-level descendant nodes for the diagnostic clues (evidence nodes), each independently linked by a conditional probability matrix to a root node containing the diagnostic alternatives (decision node). One of the evidence nodes was based on the tissue architecture and the others were based on cell features. The system was designed to be interactive, in that the histopathologist entered evidence into the network in the form of likelihood ratios for outcomes at each evidence node. The efficiency of the network was tested on a series of 110 prostate specimens, subdivided as follows: 22 cases of non-neoplastic prostate or benign prostatic tissue (NP), 22 PINs of low grade (PINlow), 22 PINs of high grade (PINhigh), 22 prostatic adenocarcinomas with cribriform pattern (PACcri), and 22 prostatic adenocarcinomas with large acinar pattern (PAClgac). The results obtained in the benign and malignant categories showed that the belief for the diagnostic alternatives is very high, the values being in general more than 0.8 and often close to 1.0. When considering the PIN lesions, the network classified and graded most of the cases with high certainty. However, there were some cases which showed values less than 0.8 (13 cases out of 44), thus indicating that there are situations in which the feature changes are intermediate between contiguous categories or grades. Discrepancy between morphological grading and the BBN results was observed in four out of 44 PIN cases: one PINlow was classified as PINhigh and three PINhigh were classified as PINlow. In conclusion, the network can grade PlN lesions and differentiate them from other prostate lesions with certainty. In particular, it offers a descriptive classifier which is readily implemented and which allows the use of linguistic, fuzzy variables.

EXPERT-SYSTEM SUPPORT USING BAYESIAN BELIEF NETWORKS IN THE DIAGNOSIS OF FINE-NEEDLE ASPIRATION BIOPSY SPECIMENS OF THE BREAST

Aim-To develop an expert system model for the diagnosis of fine needle aspiration cytology (FNAC) of the breast.

Methods-Knowledge and uncertainty were represented in the form of a Bayesian belief network which permitted the combination of diagnostic evidence in a cumulative manner and provided a final probability for the possible diagnostic outcomes. The network comprised 10 cytological features (evidence nodes), each independently linked to the diagnosis (decision node) by a conditional probability matrix. The system was designed to be interactive in that the cytopathologist entered evidence into the network in the form of likelihood ratios for the outcomes at each evidence node.

Results-The efficiency of the network was tested on a series of 40 breast FNAC specimens. The highest diagnostic probability provided by the network agreed with the cytopathologists' diagnosis in 100% of cases for the assessment of discrete, benign, and malignant aspirates. A typical probably benign cases were given probabilities in favour of a benign diagnosis. Suspicious cases tended to have similar probabilities for both diagnostic outcomes and so, correctly, could not be assigned as benign or malignant. A closer examination of cumulative belief graphs for the diagnostic sequence of each case provided insight into the diagnostic process, and quantitative data which improved the identification of suspicious cases.

Conclusion-The further development of such a system will have three important roles in breast cytodiagnosis: (1) to aid the cytologist in making a more consistent and objective diagnosis; (2) to provide a teaching tool on breast cytological diagnosis for the non-expert; and (3) it is the first stage in the development of a system capable of automated diagnosis through the use of expert system machine vision.

Limbal stem cell deficiency:Concept, aetiology, clinical presentation, diagnosis and management

Defects in renewal and repair of ocular surface as a result of limbal stem cell deficiency are now known to cause varying ocular, surface morbidity including persistent photophobia, repeated and persistent surface breakdown and overt conjunctivalisation of the cornea. Ocular conditions with abnormalities of ocular surface repair include pterygium, limbal tumours, aniridia, severe scarring following burns, cicatricial pemphigoid and Stevens-Johnson Syndrome, sequelae of mustard gas exposure and Herpes simplex epithelial disease, radiation keratopathy, contact lens induced keratopathy, neuroparalytic keratitis and drug toxicity. Restoring ocular health in these eyes has traditionally been frustrating. An understanding of these intricate cell renewal and maintenance processes has spurred the evolution in recent years of new treatment methods for several blinding diseases of the anterior segment; many more exciting modalities are in the offing. However, there is inadequate awareness among ophthalmologists about the current principles of management of ocular surface disorders. The purpose of this article is to help elucidate the important principles and current treatment methods relevant to ocular surface disorders.

Corneal Allograft Rejection:Risk Factors, Diagnosis, Prevention, and Treatment

Recent advances in corneal graft technology, including donor tissue retrieval, storage and surgical techniques, have greatly improved the clinical outcome of corneal grafts. Despite these advances, immune mediated corneal graft rejection remains the single most important cause of corneal graft failure. Several host factors have been identified as conferring a "high risk" status to the host. These include: more than two quadrant vascularisation, with associated lymphatics, which augment the afferent and efferent arc of the immune response; herpes simplex keratitis; uveitis; silicone oil keratopathy; previous failed (rejected) grafts; "hot eyes"; young recipient age; and multiple surgical procedures at the time of grafting. Large grafts, by virtue of being closer to the host limbus, with its complement of vessels and antigen-presenting Langerhans cells, also are more susceptible to rejection. The diagnosis of graft rejection is entirely clinical and in its early stages the clinical signs could be subtle. Graft rejection is largely mediated by the major histocompatibility antigens, minor antigens and perhaps blood group ABO antigens and some cornea-specific antigens. Just as rejection is mediated by active immune mediated events, the lack of rejection (tolerance) is also sustained by active immune regulatory mechanisms. The anterior chamber associated immune deviation (ACAID) and probably, conjunctiva associated lymphoid tissue (CALT) induced mucosal tolerance, besides others, play an important role. Although graft rejection can lead to graft failure, most rejections can be readily controlled if appropriate management is commenced at the proper time. Topical steroids are the mainstay of graft rejection management. In the high-risk situations however, systemic steroids, and other immunosuppressive drugs such as cyclosporin and tacrolimus (FK506) are of proven benefit, both for treatment and prevention of rejection.