Standard setting anchor statements: a double cross-over trial of two different methods.

This article was migrated. The article was marked as recommended. Context: We challenge the philosophical acceptability of the Angoff method, and propose an alternative method of standard setting based on how important it is for candidates to know the material each test item assesses, and not how difficult it is for a subgroup of candidates to answer each item. Methods: The practicalities of an alternative method of standard setting are evaluated here, for the first time, with direct comparison to an Angoff method. To negate bias due to any leading effects, a prospective cross-over design was adopted involving two groups of judges (n=7 and n=8), both of which set the standards for the same two 100 item multiple choice question tests, by the two different methods. Results: Overall, we found that the two methods took a similar amount of time to complete. The alternative method produced a higher cut-score (by 12-14%), and had a higher degree of variability between judges' cut-scores (by 5%). When using the alternative method, judges reported a small, but statistically significant, increase in their confidence to decide accurately the standard (by 3%). Conclusion: This is a new approach to standard setting where the quantitative differences are slight, but there are clear qualitative advantages associated with use of the alternative method.

Timing is key to providing modified assessments for students with specific learning difficulties.

INTRODUCTION: Medical students who are diagnosed with a specific learning difficulty (SpLD) will typically receive a reasonable adjustment within examinations in the form of modified assessment provision (MAP). This study investigated whether the timing of SpLD diagnosis and subsequent implementation of MAP has an impact on performance in applied medical knowledge multiple choice question (MCQ) assessments. METHOD: The MCQ performance of 108 students diagnosed with SpLD who received a MAP was monitored and compared with 1960 students who received an unmodified assessment, over 5 years of a medical program. Students who received a SpLD diagnosis in the latter years of the program were identified as not receiving a MAP in assessments prior to diagnosis. RESULTS: Differences were found between declaration and diagnosis, with 44.4% of students who declared and 48.1% who did not declare subsequently receiving a diagnosis. Students with SpLD who receive a MAP increase their applied medical knowledge assessment performance, although there is a delay of up to a year for this impact to reach significance. CONCLUSION: Early diagnosis of SpLD is necessary to ensure the intended benefit is received from MAP.

Totally implanted venous access-associated adverse events in oncology: Results from a prospective 1-year surveillance programme.

INTRODUCTION: During the last decade, most studies on totally implanted venous access-associated adverse events (TIVA-AE) were conducted retrospectively and/or were based on a limited sample size. The aim of our survey was two-fold: to estimate the incidence of TIVA-AE and to identify risk factors in patients with cancer. METHODS: Data from our routine surveillance of TIVA-AE were collected prospectively between October 2009 and January 2011 in two oncology referral centers in Northern France. The open cohort under surveillance during the same time period was reconstituted retrospectively using data from the hospital information systems. Incidences of first TIVA-AE per 1000 TIVA-days were calculated. Risk factors were identified using multivariate logistic regressions. RESULTS: We included 2286 cancer patients, corresponding to 582,347 TIVA-days. Among the 133 first TIVA-AE observed (incidence 0.23 per 1000 TIVA-days [0.19-0.27]), there were 50 infectious AE (incidence 0.09 [0.06-0.11]) and 83 non-infectious AE (incidence 0.14 [0.11-0.17]). Compared to non-metastatic solid cancers, metastatic cancers (aOR=2.3 [0.9-6.0]), and hematologic malignancies (aOR=3.2 [1.1-8.8]) tended to be associated with a higher risk of infectious TIVA-AE (P=0.087). Solid cancer type was associated with non-infectious TIVA-AE (P=0.030), especially digestive cancers. DISCUSSION: We report accurate estimations of TIVA-AE incidences in one of the largest populations among previously published studies. As in previous studies, metastatic cancers and hematologic malignancies tended to be associated with a higher risk of infectious TIVA-AE. Further studies are warranted to confirm the effect of digestive cancers.

Morbidity-mortality conference for adverse events associated with totally implanted venous access for cancer chemotherapy.

PURPOSE: Although considered safer than central venous catheters for administration of cancer chemotherapy, totally implanted venous access (TIVA) is associated with adverse events that may impair prognosis and quality of life of patients receiving chemotherapy. Our aim was to assess the feasibility and interest of surveillance of cancer chemotherapy TIVA-adverse events (AE), associated with morbidity-mortality conferences (MMCs) on TIVA-AE. METHODS: We performed a prospective interventional study in two hospitals (a university hospital and a comprehensive care center). For each cancer chemotherapy care pathway within each hospital, we set up surveillance of TIVA-AE and MMC on these events. Patients included in surveillance were those with a TIVA either placed or used for chemotherapy cycles in one of the participating wards. Feasibility of MMC was assessed by the number of MMC meetings that actually took place and the number of participants at each meeting. The interest of MMC was assessed by the number of TIVA-AE identified and analyzed, and the number and type of improvement actions selected and actually implemented. RESULTS: We recorded 0.41 adverse events per 1000 TIVA-day. MMCs were implemented in all care pathways, with sustained pluriprofessional attendance throughout the survey; 39 improvement actions were identified during meetings, and 18 were actually implemented. CONCLUSIONS: Surveillance of TIVA-AE associated with MMC is feasible and helps change practices. It could be useful for improving care of patients undergoing cancer chemotherapy.

Features of Blastocystis spp. in xenic culture revealed by deconvolutional microscopy.

Blastocystis spp. are common human enteric parasites with complex morphology and have been reported to cause irritable bowel syndrome (IBS). Deconvolutional microscopy with time-lapse imaging and fluorescent spectroscopy of xenic cultures of Blastocystis spp. from stool samples of IBS patients and from asymptomatic, healthy pigs allowed observations of living organisms in their natural microbial environment. Blastocystis organisms of the vacuolated, granular, amoebic and cystic forms were observed to autofluorescence in the 557/576 emission spectra. Autofluorescence could be distinguished from fluorescein-conjugated Blastocystis-specific antibody labelling in vacuolated and granular forms. This antibody labelled Blastocystis subtypes 1, 3 and 4 but not 5. Surface pores of 1 µm in diameter were observed cyclically opening and closing over 24 h. Vacuolated forms extruded a viscous material from a single surface point with coincident deflation that may demonstrate osmoregulation. Tear-shaped granules were observed exiting from the surface of an amoebic form, but their origin and identity remain unknown.

Invasive aspergillosis in neutropenic patients during hospital renovation: effectiveness of mechanical preventive measures in a prospective cohort of 438 patients.

BACKGROUND: Aspergillus species are the main cause of invasive fungal disease for patients with severe and prolonged neutropenia. Building or renovation works have been shown as one of the major causes of outbreaks of aspergillosis. OBJECTIVES: This study aimed to assess the effectiveness of introduction and adaptation by air sampling of mechanical preventive measures on the incidence of invasive pulmonary aspergillosis in neutropenic patients during hospital renovation. PATIENTS: All of the patients admitted for prolonged and severe neutropenia during a renovation period from 2003 to 2008 were prospectively enrolled. Invasive pulmonary aspergillosis (IPA) cases were classified as possible, probable, and proven, according to the 2008 European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group criteria. The effectiveness of preventive measures was determined by air sampling. RESULTS: We recorded 705 hospitalizations for neutropenia concerning 438 patients. The majority of hospitalized neutropenic patients was treated for acute leukemia (38.3 %), followed by patients suffering from non-Hodgkin and Hodgkin lymphomas (33 %). The total cumulative incidence of probable and proven IPA was 4.1 %. Risk factors for developing IPA were underlying disease, treatment course at the time of hospitalization, and the mean duration of hospitalization and of neutropenia. CONCLUSIONS: In this prospective study, the incidence of invasive pulmonary aspergillosis did not increase in neutropenic patients during a renovation period because of efficient mechanical preventive measures systematically adjusted using the results of air sampling.

Anthelminthic activity of the cyclotides (kalata B1 and B2) against schistosome parasites.

The risk of reduced sensitivity of the human schistosomes to praziquantel has led to efforts to find new therapies. Here, the cyclotides kalata B1 (kB1), kalata B2 (kB2), MCoCC-1, and MCoTI-II, cyclic peptides extracted from plants and shown to be potent against nematodes and insects, were tested for antischistosome activity. In vitro assays showed that high concentrations (500-1000 µg/mL) of either kB1 or kB2 killed Schistosoma japonicum and Schistosoma mansoni adults within 5 min, whereas MCoTI-II and MCoCC-1 had no effect. Lethal concentrations to kill 50% of the population for kB2 was 15.5 ± 7.4 µg/mL at 1 h for male S. japonicum (Philippine strain). Males were more susceptible than females. kB2 showed higher antischistosome activity than kB1 and killing time was concentration-dependent. Mode of action studies revealed that kB1 and 2 lysed the tegument of adult worms. Lysis of myofibrils was not demonstrated, but longitudinal and radial muscle fibers were distorted, an observation consistent with strong coiling of the parasites after drug exposure. A single dose of kB2 administered either orally or intravenously, reduced worm burdens in S. japonicum-infected mice from 15% to 60%. However, treatment of S. mansoni-infected mice did not result in reduction in worm burdens. Our studies show that kB2 acts as a promising antischistosomal against Philippine S. japonicum, and it or other cyclotides may be developed further as general anthelminthics. With thousands of cyclotides predicted to occur in plants, and the amenability of these peptides to combinatorial variation, there is potential for their exploitation as wide-spectrum anthelminthics.

Real-time PCR as a surveillance tool for the detection of Trichinella infection in muscle samples from wildlife.

Trichinella nematodes are the causative agent of trichinellosis, a meat-borne zoonosis acquired by consuming undercooked, infected meat. Although most human infections are sourced from the domestic environment, the majority of Trichinella parasites circulate in the natural environment in carnivorous and scavenging wildlife. Surveillance using reliable and accurate diagnostic tools to detect Trichinella parasites in wildlife hosts is necessary to evaluate the prevalence and risk of transmission from wildlife to humans. Real-time PCR assays have previously been developed for the detection of European Trichinella species in commercial pork and wild fox muscle samples. We have expanded on the use of real-time PCR in Trichinella detection by developing an improved extraction method and SYBR green assay that detects all known Trichinella species in muscle samples from a greater variety of wildlife. We simulated low-level Trichinella infections in wild pig, fox, saltwater crocodile, wild cat and a native Australian marsupial using Trichinella pseudospiralis or Trichinella papuae ethanol-fixed larvae. Trichinella-specific primers targeted a conserved region of the small subunit of the ribosomal RNA and were tested for specificity against host and other parasite genomic DNAs. The analytical sensitivity of the assay was at least 100 fg using pure genomic T. pseudospiralis DNA serially diluted in water. The diagnostic sensitivity of the assay was evaluated by spiking 10 g of each host muscle with T. pseudospiralis or T. papuae larvae at representative infections of 1.0, 0.5 and 0.1 larvae per gram, and shown to detect larvae at the lowest infection rate. A field sample evaluation on naturally infected muscle samples of wild pigs and Tasmanian devils showed complete agreement with the EU reference artificial digestion method (k-value=1.00). Positive amplification of mouse tissue experimentally infected with T. spiralis indicated the assay could also be used on encapsulated species in situ. This real-time PCR assay offers an alternative highly specific and sensitive diagnostic method for use in Trichinella wildlife surveillance and could be adapted to wildlife hosts of any region.

Successful treatment of disseminated fusariosis with voriconazole in an acute lymphoblastic leukaemia patient.

Fusarium species are actually the second most common pathogenic mould in immunocompromised patients, and it is difficult to treat such fusarial infections with current antifungal agents. We report the case of a 53-year-old woman with Philadelphia-positive acute lymphoblastic leukaemia. During induction chemotherapy with febrile neutropenia, she developed a disseminated fusariosis, with persistent fever refractory to antibacterial agents and caspofungin (as empirical therapy), painful skin lesions and respiratory impairment. Fusarium solani was isolated from skin biopsy. Voriconazole was successfully implemented as antifungal curative therapy. During the second intensive chemotherapy no reactivation of fusariosis was detected.

Efficacy of toltrazuril 5 % suspension against Eimeria bovis and Eimeria zuernii in calves and observations on the associated immunopathology.

16 Calves were each infected with suspensions containing a mixture of approximately 230,000 Eimeria bovis and 70,000 E. zuernii oocysts, which resulted in detection of oocysts in faeces of 12 of 16 calves by day +14 after infection. On day +14 after infection calves were either treated (n = 8) with toltrazuril at 15 mg/kg body weight or with a placebo. Observations were made on the clinical condition, faecal score and liveweight of calves daily from one day post infection (pi) until 24 days pi when all calves were euthanised and examined post mortem. Samples were collected from ileum and colon for histological, immunohistochemical and gene expression studies. The study demonstrated an efficacy of toltrazuril for the treatment of E. bovis and E. zuernii infections in calves reaching 99 % (based on arithmetic mean oocyst counts in faeces) within three days of treatment and remaining at or above this level for six days. Toltrazuril did not have a significant effect on the pattern and extent of immune cellular infiltration in the mucosa of ileum and colon, but the expression of the genes coding IL-2, IL-10 and TNF-α in the ileum and TNF-α in the colon were elevated in calves treated with toltrazuril. Higher levels of oocyst shedding were significantly associated with lower expression of genes coding for IL-2, IL-10 and higher IP-10. It is concluded that toltrazuril is effective for the treatment of coccidiosis due to E. bovis and E. zuernii in calves and enables the development of a normal or enhanced immune response to infection.

Identification of immune genes and proteins involved in the response of bovine mammary tissue to Staphylococcus aureus infection.

BACKGROUND: Mastitis in dairy cattle results from infection of mammary tissue by a range of micro-organisms but principally coliform bacteria and Gram positive bacteria such as Staphylococcus aureus. The former species are often acquired by environmental contamination while S. aureus is particularly problematic due to its resistance to antibiotic treatments and ability to reside within mammary tissue in a chronic, subclinical state. The transcriptional responses within bovine mammary epithelial tissue subjected to intramammary challenge with S. aureus are poorly characterised, particularly at the earliest stages of infection. Moreover, the effect of infection on the presence of bioactive innate immune proteins in milk is also unclear. The nature of these responses may determine the susceptibility of the tissue and its ability to resolve the infection. RESULTS: Transcriptional profiling was employed to measure changes in gene expression occurring in bovine mammary tissues sampled from three dairy cows after brief and graded intramammary challenges with S. aureus. These limited challenges had no significant effect on the expression pattern of the gene encoding beta-casein but caused coordinated up-regulation of a number of cytokines and chemokines involved in pro-inflammatory responses. In addition, the enhanced expression of two genes, S100 calcium-binding protein A12 (S100A12) and Pentraxin-3 (PTX3) corresponded with significantly increased levels of their proteins in milk from infected udders. Both genes were shown to be expressed by mammary epithelial cells grown in culture after stimulation with lipopolysaccharide. There was also a strong correlation between somatic cell count, a widely used measure of mastitis, and the level of S100A12 in milk from a herd of dairy cows. Recombinant S100A12 inhibited growth of Escherichia coli in vitro and recombinant PTX3 bound to E. coli as well as C1q, a subunit of the first component of the complement cascade. CONCLUSION: The transcriptional responses in infected bovine mammary tissue, even at low doses of bacteria and short periods of infection, probably reflect the combined contributions of gene expression changes resulting from the activation of mammary epithelial cells and infiltrating immune cells. The secretion of a number of proinflammatory cytokines and chemokines from mammary epithelial cells stimulated by the bacteria serves to trigger the recruitment and activation of neutrophils in mammary tissue. The presence of S100A12 and PTX3 in milk from infected udder quarters may increase the anti-bacterial properties of milk thereby helping to resolve the mammary tissue infection as well as potentially contributing to the maturation of the newborn calf epithelium and establishment of the newborn gut microbial population.

15-Deoxy-Delta12,14-prostaglandin J2 induces chemokine expression, oxidative stress and microfilament reorganization in bovine mammary epithelial cells.

The roles of the pro-adipogenic ligands of the transcription factor Peroxisome Proliferator Activated Receptor gamma (PPARG) in regulating innate immune responses in bovine mammary epithelial cells (bMEC) were investigated using quantitative real-time PCR. The analyses revealed that 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) enhanced the expression of Interleukin 8 (IL-8) and Chemokine (C-X-C motif) ligand 6 (CXCL6) in these cells in a dose-dependent manner. 15d-PGJ2 also induced the expression of transcripts encoding proteins involved in oxidative stress, including Ferritin heavy chain and Superoxide dismutase 1, as well as substantial microfilament reorganization. In contrast, synthetic PPARG agonists displayed a different and much smaller range of effects on the cells, causing down-regulation of Interleukin 1-beta, Interleukin 6 and IL-8 and increased expression of Chemokine (C-C motif) ligand 2 (CCL2) and Tumour necrosis factor alpha (TNFalpha). In an independent analysis, the cells were pre-incubated with PPARG agonists followed by lipopolysaccharide stimulation. This study revealed that troglitazone increased the responsiveness of the cells to lipopolysaccharide resulting in up-regulation of Interleukin 1-beta, TNFalpha, IL-8, CCL2 and CXCL6 while 15d-PGJ2 caused down-regulation of TNFalpha, CCL2 and CXCL6. These findings are relevant to understanding the anti-inflammatory potential of the PPARG ligands and underline different mechanisms of action of 15d-PGJ2 and troglitazone in bMEC. Furthermore, the present results demonstrate that the generation of pro-inflammatory mediators can be modulated by currently available therapeutic agents and may therefore be of value in the treatment of mastitis in ruminants.

Construction and validation of a Bovine Innate Immune Microarray.

BACKGROUND: Microarray transcript profiling has the potential to illuminate the molecular processes that are involved in the responses of cattle to disease challenges. This knowledge may allow the development of strategies that exploit these genes to enhance resistance to disease in an individual or animal population. RESULTS: The Bovine Innate Immune Microarray developed in this study consists of 1480 characterised genes identified by literature searches, 31 positive and negative control elements and 5376 cDNAs derived from subtracted and normalised libraries. The cDNA libraries were produced from 'challenged' bovine epithelial and leukocyte cells. The microarray was found to have a limit of detection of 1 pg/microg of total RNA and a mean slide-to-slide correlation co-efficient of 0.88. The profiles of differentially expressed genes from Concanavalin A (ConA) stimulated bovine peripheral blood lymphocytes were determined. Three distinct profiles highlighted 19 genes that were rapidly up-regulated within 30 minutes and returned to basal levels by 24 h; 76 genes that were up-regulated between 2-8 hours and sustained high levels of expression until 24 h and 10 genes that were down-regulated. Quantitative real-time RT-PCR on selected genes was used to confirm the results from the microarray analysis. The results indicate that there is a dynamic process involving gene activation and regulatory mechanisms re-establishing homeostasis in the ConA activated lymphocytes. The Bovine Innate Immune Microarray was also used to determine the cross-species hybridisation capabilities of an ovine PBL sample. CONCLUSION: The Bovine Innate Immune Microarray has been developed which contains a set of well-characterised genes and anonymous cDNAs from a number of different bovine cell types. The microarray can be used to determine the gene expression profiles underlying innate immune responses in cattle and sheep.

Lipopolysaccharide and lipoteichoic acid induce different innate immune responses in bovine mammary epithelial cells.

The objective of the present study was to characterize the innate immune responses induced by in vitro stimulation of bovine primary mammary epithelial cells (bMEC) using gram-negative lipopolysaccharide (LPS) and gram-positive lipoteichoic acid (LTA) bacterial cell wall components. Quantitative real-time PCR (qRT-PCR) was employed to examine the mRNA expression of a panel of 22 cytokines, chemokines, beta-defensins and components of the Toll-Like Receptor signaling pathway. Stimulation of bMEC with LPS for 24h elicited a marked increase in mRNA expression for IL-1beta, IL-8, TNFalpha, CXCL6 and beta-defensin while members of the Toll-Like Receptor pathway, although present, were largely unaffected. Surprisingly, stimulation of these cells with LTA for 24 h did not significantly alter the expression of these genes. A time course of the expression of IL-1beta, IL-8, TNFalpha, CXCL6 and beta-defensin was subsequently performed. The mRNA levels of all genes increased rapidly after stimulation for 2-4 h with both LPS and LTA but only the former treatment resulted in sustained responses. In contrast, the increased gene expression for LTA stimulated cells returned to resting levels after 8-16 h with the exception of beta-defensin, which remained up-regulated. The limited and unsustained cytokine response to LTA may explain why mastitis caused by gram-positive bacteria has greater potential for chronic intra-mammary infection than gram-negative infection. It was concluded that bovine mammary epithelial cells have a strong but differential capacity to mount innate immune responses to bacterial cell wall components.

Association of increased levels of heavy-chain ferritin with increased CD4+ CD25+ regulatory T-cell levels in patients with melanoma.

We have shown previously that melanoma cells in culture release heavy-chain ferritin (H-Ferritin) into supernatants and that this is responsible for the suppression of responses of peripheral blood lymphocytes stimulated by anti-CD3. These effects were mediated by activation of regulatory T cells to produce interleukin (IL)-10. In the present study, we examined whether a similar relation might exist between levels of H-Ferritin and activation of regulatory T cells in patients with melanoma. Ferritin levels were evaluated by ELISA and regulatory T-cell numbers were assessed by three-color flow cytometry to identify CD4(+) CD25(+) CD69(-) T cells. CD69 positive cells were excluded to avoid inclusion of normal activated CD4, CD25 expressing T cells. Measurements of H- and light-chain (L)-Ferritin by ELISA revealed that H- but not L-Ferritin was elevated in the circulation of melanoma patients. In addition, these studies revealed a marked increase in the number of CD4(+) CD25(+) CD69(-) T cells in such patients, compared with age-matched controls. The ratio of H-Ferritin:L-Ferritin correlated with the levels of regulatory T cells consistent with a causal relation between unbound H-Ferritin levels and the activation of regulatory T cells. H-Ferritin or regulatory T cells did not, however, correlate with the stage of the melanoma. These results provide evidence for the importance of H-Ferritin in the induction of regulatory T cells in patients with melanoma and provide additional insight into the suppression of immune responses in such patients.

Heavy chain ferritin activates regulatory T cells by induction of changes in dendritic cells.

Heavy chain ferritin (H-ferritin) is a component of the iron-binding protein, ferritin. We have previously shown that H-ferritin inhibits anti-CD3-stimulated lymphocyte proliferation and that this was due to increased production of interleukin-10 (IL-10). In the present study we have shown that induction of IL-10 production was due to effects of H-ferritin on adherent antigen-presenting cells (APCs) in blood and monocyte-derived dendritic cells (MoDCs). IL-10 was produced by a subpopulation of CD4 T cells, which expressed the CD25 component of the IL-2 receptor and the CTLA-4 receptor characteristic of regulatory T cells. The changes induced in MoDCs were compared with those induced by CD40L and their significance tested by inhibition with monoclonal antibodies. These studies indicated that H-ferritin induced relatively greater expression of CD86 and B7-H1 on MoDCs and that monoclonal antibodies against their receptors, CTLA-4 and programmed death receptor-1 (PD-1), inhibited IL-10 production from the regulatory T cells. H-ferritin did not appear to induce direct production of the cytokines IL-2, IL-4, IL-6, IL-10, IL-12, or interferon-gamma from the DCs. These results are consistent with the thesis that H-ferritin induces B7-H1 and CD86 (B7-2) on APCs, which in turn induce IL-10 production from regulatory T cells. This is possibly one mechanism by which melanoma cells may induce changes in APCs in the vicinity of the tumor and result in suppression of immune responses by induction of regulatory T cells.