Characterization of Haemonchus contortus P-glycoprotein-16 and its interaction with the macrocyclic lactone anthelmintics.

Anthelmintic resistance in veterinary nematodes, including Haemonchus contortus, has become a limitation to maintaining high standards of animal health. Resistance in this parasite, to all drug families including the macrocyclic lactones (MLs) is a serious issue worldwide. Mechanisms of resistance to the MLs appear to be complex and to include the elimination of these compounds by ABC transporter-like proteins present in nematodes. In order to investigate the potential involvement of ABC transporters in ML resistance in H. contortus, we have characterized the functionality of the ABC transporter H. contortus P-glycoprotein-16 (Hco-PGP-16) expressed in mammalian cells. This has included a study of its interaction with different MLs, including the avermectins, abamectin (ABA) and ivermectin (IVM), and the milbemycin, moxidectin (MOX). Hco-PGP-16 transport activity was studied using the fluorophore Rhodamine 123 (Rho 123). Transfected cells expressing Hco-PGP-16 accumulated less than 50% of Rho 123 than control cells, suggesting an active transport of this tracer dye by Hco-PGP-16. The influence of the MLs on the Rho123 transport by Hco-PGP-16 was then investigated. A marked inhibition of Rho123 transport by ABA and IVM was observed. In contrast, MOX showed less effect on inhibition of Rho123 transport by Hco-PGP-16, and the inhibition was not saturable. The difference in the interaction of the avermectins and MOX with Hco-PGP-16 may help explain the slower rate of development of resistance to MOX compared with the avermectins in H. contortus.

Molecular and morphological characterization of the tapeworm Taenia hydatigena (Pallas, 1766) in sheep from Iran.

Although Taenia hydatigena is one of the most prevalent taeniid species of livestock, very little molecular genetic information exists for this parasite. Up to 100 sheep isolates of T. hydatigena were collected from 19 abattoirs located in the provinces of Tehran, Alborz and Kerman. A calibrated microscope was used to measure the larval rostellar hook lengths. Following DNA extraction, fragments of cytochrome c oxidase 1 (CO1) and 12S rRNA genes were amplified by the polymerase chain reaction method and the amplicons were subjected to sequencing. The mean total length of large and small hooks was 203.4 µm and 135.9 µm, respectively. Forty CO1 and 39 12S rRNA sequence haplotypes were obtained in the study. The levels of pairwise nucleotide variation between individual haplotypes of CO1 and 12S rRNA genes were determined to be between 0.3-3.4% and 0.2-2.1%, respectively. The overall nucleotide variation among all the CO1 haplotypes was 9.7%, and for all the 12S rRNA haplotypes it was 10.1%. A significant difference was observed between rostellar hook morphometry and both CO1 and 12S rRNA sequence variability. A significantly high level of genetic variation was observed in the present study. The results showed that the 12S rRNA gene is more variable than CO1.

Gene-expression differences in peripheral blood between lithium responders and non-responders in the Lithium Treatment-Moderate dose Use Study (LiTMUS).

This study was designed to identify genes whose expression in peripheral blood may serve as early markers for treatment response to lithium (Li) in patients with bipolar disorder. Although changes in peripheral blood gene-expression may not relate directly to mood symptoms, differences in treatment response at the biochemical level may underlie some of the heterogeneity in clinical response to Li. Subjects were randomized to treatment with (n=28) or without (n=32) Li. Peripheral blood gene-expression was measured before and 1 month after treatment initiation, and treatment response was assessed after 6 months. In subjects treated with Li, 62 genes were differentially regulated in treatment responders and non-responders. Of these, BCL2L1 showed the greatest difference between Li responders and non-responders. These changes were specific to Li responders (n=9), and were not seen in Li non-responders or patients treated without Li, suggesting that they may have specific roles in treatment response to Li.

Anthelmintic resistance: markers for resistance, or susceptibility?

The Consortium for Anthelmintic Resistance and Susceptibility (CARS) brings together researchers worldwide, with a focus of advancing knowledge of resistance and providing information on detection methods and treatment strategies. Advances in this field suggest mechanisms and features of resistance that are shared among different classes of anthelmintic. Benzimidazole resistance is characterized by specific amino acid substitutions in beta-tubulin. If present, these substitutions increase in frequency upon drug treatment and lead to treatment failure. In the laboratory, sequence substitutions in ion-channels can contribute to macrocyclic lactone resistance, but there is little evidence that they are significant in the field. Changes in gene expression are associated with resistance to several different classes of anthelmintic. Increased P-glycoprotein expression may prevent drug access to its site of action. Decreased expression of ion-channel subunits and the loss of specific receptors may remove the drug target. Tools for the identification and genetic analysis of parasitic nematodes and a new online database will help to coordinate research efforts in this area. Resistance may result from a loss of sensitivity as well as the appearance of resistance. A focus on the presence of anthelmintic susceptibility may be as important as the detection of resistance.

Population genetics of anthelmintic resistance in parasitic nematodes.

A key aim of anthelmintic resistance research is to identify molecular markers that could form the basis of sensitive and accurate diagnostic tests. These would provide powerful tools to study the origin and spread of anthelmintic resistance in the field and to monitor strategies aimed at preventing and managing resistance. Molecular markers could also form the basis of routine diagnostic tests for use in surveillance and clinical veterinary practice. Much of the research conducted to date has focused on the investigation of possible associations of particular candidate genes with the resistance phenotype. In the future, as full parasite genome sequences become available, there will be an opportunity to apply genome-wide approaches to identify the genetic loci that underlie anthelmintic resistance. Both the interpretation of candidate gene studies and the application of genome-wide approaches require a good understanding of the genetics and population biology of the relevant parasites as well as knowledge of how resistance mutations arise and are selected in populations. Unfortunately, much of this information is lacking for parasitic nematodes. This review deals with a number of aspects of genetics and population biology that are pertinent to these issues. We discuss the possible origins of resistance mutations and the likely effects of subsequent selection on the genetic variation at the resistance-conferring locus. We also review some of the experimental approaches that have been used to test associations between candidate genes and anthelmintic resistance phenotypes and highlight implications for future genome-wide studies.

Expression of ezrin in glial tubes in the adult subventricular zone and rostral migratory stream.

Ezrin is a member of the ERM (ezrin-radixin-moesin) family of membrane-cytoskeletal linking proteins. ERM proteins are involved in a wide variety of cellular functions including cell motility, signal transduction, cell-cell interaction and cell-matrix recognition. A recent in situ hybridization study showed that the mRNA encoding ezrin is expressed in neurogenic regions of the mature brain including the subventricular zone (SVZ) and rostral migratory stream (RMS); however, the specific cell types expressing ezrin and their relationship to migrating and proliferating cells in these regions have not been characterized previously. In this study, we used immunocytochemistry to perform double labeling with a variety of cell-type specific markers to characterize the expression of ezrin in the SVZ and RMS of adult mice. Ezrin was expressed at high levels in both the SVZ and RMS where ezrin-immunopositive processes formed a trabecular network surrounding the proliferating and migrating cells. Ezrin-positive cells co-labeled with the glial makers S100beta and GFAP (glial fibrillary acidic protein), but only minimally with the early neuronal markers beta III tubulin and polysialylated form of neural cell adhesion molecule 1 (PSA-NCAM), indicating that ezrin was expressed primarily in the glial tube cells. Ezrin positive cells also expressed beta-catenin, a membrane-complex protein previously implicated in the regulation of stem-cell proliferation and neuronal migration. Glial tube cells act as both precursors of, and a physical channel for, migrating neuroblasts. Bi-directional signals between glial tube cells and migrating neuroblasts have been shown to regulate the rates of both proliferation of the precursor cells and migration of the newly generated neuroblasts. Our finding that ezrin and beta-catenin are both present at the cell membrane of the glial tube cells suggests that these proteins may be involved in those signaling processes.

Ivermectin selection on beta-tubulin: evidence in Onchocerca volvulus and Haemonchus contortus.

Ivermectin resistance is common in trichostrongylid nematodes of livestock, such as Haemonchus contortus. This anthelmintic is the only drug approved for mass administration to control onchocerciasis caused by the nematode parasite, Onchocerca volvulus. In parts of West Africa up to 18 rounds of ivermectin treatment have been administered to communities and there are reports of poor parasitological responses to treatment. Understanding ivermectin resistance and ivermectin selection is an important step to reduce selection pressure for resistance, and to develop molecular markers which can be used to monitor the development of resistance and its spread. Here we report evidence that ivermectin selection changes the frequency of beta-tubulin alleles in both the sheep parasite, H. contortus, and the human parasite, O. volvulus. In O. volvulus we have been able to look at the frequency of beta-tubulin alleles in O. volvulus obtained before any ivermectin was used in humans in Africa, and following its widespread use. In H. contortus, we have been able to look at the frequency of beta-tubulin alleles in a strain which has not seen any anthelmintic selection and in an ivermectin selected strain derived from the unselected strain. We have found ivermectin selects on beta-tubulin in both of these nematode species. In the case of O. volvulus, we had previously reported that ivermectin selects for specific single nucleotide polymorphisms in the O. volvulus beta-tubulin gene. This polymorphism results in three amino acid changes in the H3 helix of beta-tubulin, as well as deletions in an associated intron. We report a simple PCR assay to detect the amplicon length polymorphism, resulting from these intronic deletions, which can be used to monitor the frequency of the beta-tubulin allele selected for by ivermectin in O. volvulus.

The development and use of a method to compare the costs of acute stroke across Europe.

BACKGROUND: there is a need for more information on the costs of different ways of managing stroke. Methods to compare the costs of stroke care in different countries have not been previously developed. OBJECTIVE: to develop and use a method to compare the costs of acute stroke care across Europe. SETTING: acute hospitals in 13 different European centres. SUBJECTS AND METHODS: we included in the study stroke patients hospitalized during 1996-7 at 13 centres across Europe (n=2072). We recorded the duration of acute hospital stay and use of investigations. Mean costs for each centre were predicted using linear regression analysis to adjust for case-mix differences. RESULTS: the average acute hospital stay ranged from 9 days (Spain) to 35 days (UK; P < 0.001). The predicted mean cost of treating conscious, continent men aged > 74 ranged from $220 (95% confidence interval 191-254) in Latvia to $5164 (4294-6191) in Austria. CONCLUSIONS: differences in the acute costs of stroke exist across Europe because of differences in clinical practice and unit costs. This methodology will be used to capture the costs incurred by a broad range of care providers. These estimates will then be suitable for using in cost-effectiveness analysis.

Genetic variability determinants of Helicobacter pylori: influence of clinical background and geographic origin of isolates.

Helicobacter pylori has an unusual pattern of genetic variation, which complicates research on this organism. To gain a better understanding of the forces behind this phenomenon, the extent to which recombination and single point mutations affect genetic variability in H. pylori was quantified and the influence of both geographical distance and clinical background were assessed. Site-directed restriction-endonuclease digestion of 2 gene fragments was performed on 168 isolates from Montreal and Berlin. Allelic diversity was found to be much higher for H. pylori than for other bacterial species. This finding is consistent with those of previous studies on H. pylori that were conducted using other techniques. However, nucleotide diversity was within the range reported for other bacterial species. Phylogenetic analysis found no grouping of strains with clinical background or geographical origin. Recombination at a rate that resulted in linkage equilibrium within genes can explain these observations.

Is Helicobacter pylori eradication associated with gastroesophageal reflux disease?

OBJECTIVES: A recent report has suggested an association between Helicobacter pylori eradication and the development of gastroesophageal reflux disease (GERD). We therefore assessed the incidence of GERD among comparable patients having undergone successful versus failed H. pylori eradication in a controlled trial. We also compared the H. pylori strains in the subjects that developed GERD to those that did not. METHODS: Patients with a history of proven duodenal ulcer and H. pylori infection were randomised into a H. pylori eradication study. Patients subsequently underwent gastroscopy with gastric biopsies every 3 months for 1 yr. At each visit, the presence of GERD symptoms and endoscopic esophagitis were noted, and the incidence of these variables among patients in whom H. pylori eradication was successful was compared to those in whom it was not. In a subgroup, the presence of the cagA, cagE, and vacA genotypes and of cagA antibodies were determined. RESULTS: Of 98 patients randomized into this study, 11 dropped out before determination of H. pylori eradication, leaving 87 patients with analyzable results. H. pylori eradication was successful in 63 (72%). By the end of the follow-up period, patients with GERD symptoms or endoscopic esophagitis were more prevalent in the successful than in the failed eradication group (37% [95% CI: 25-50%] vs 13% [95% CI: 3-32%], p = 0.04, 95% CI for the difference: 6-42%), as were patients with GERD symptoms alone (29% [95% CI: 18-41%] vs 8% [95% CI: 1-27%], p = 0.04, 95% CI for the difference: 4-36%) or esophagitis alone (21% [95% CI: 12-33%] vs 4% [95% CI: 0-21%], p = 0.10, 95% CI for the difference: 4-29%, respectively). Multivariate analysis revealed no significant association between the incidence of symptoms or esophagitis and age, gender, Quetelet index, caffeine or alcohol intake, smoking, weight change, or the presence of a hiatus hernia. There were also no differences in the prevalence of H. pylori genotypes from patients who developed GERD as compared to those who did not. CONCLUSIONS: In this patient population, the incidence of new GERD-type symptoms or endoscopic esophagitis was greater in patients in whom successful eradication was achieved. This difference does not appear to be attributable to weight gain, habits, or specific H. pylori strains.

cagE is a virulence factor associated with Helicobacter pylori-induced duodenal ulceration in children.

This study was undertaken to determine whether infection with Helicobacter pylori strains that contain the cagE gene was associated with duodenal ulceration in children. The presence of flaA, cagA, and cagE genes was determined by polymerase chain reaction in H. pylori previously cultured from 29 children. Twelve (92%) of 13 children with duodenal ulcers were infected with cagE-positive isolates, compared with only 5 (31%) of 16 with gastritis alone (P<.01). Infection of gastric cells in tissue culture by cagE-positive H. pylori resulted in greater increments in interleukin-8 levels compared with cagE-negative strains (2.3+/-0.1 vs. 1.3+/-0.2 ng/mL in AGS cells [P<.005]; 1.5+/-0.3 vs. 0.5+/-0.2 ng/mL in KATO-III cells [P<.05]). H. pylori-containing cagE was associated with the presence of duodenal ulceration in children. Enhanced chemokine production after infection with cagE-positive H. pylori could affect disease outcome.

Association of Helicobacter pylori genotype with gastroesophageal reflux disease and other upper gastrointestinal diseases.

OBJECTIVE: Helicobacter pylori (H. pylori) is a recognized pathogen, but it may also have a protective effect for gastroesophageal reflux disease (GERD). We compared the prevalence of potential virulence factors (cagA, cagE, vacA genotypes) in GERD to other upper gastrointestinal diseases and controls. METHODS: A total of 405 patients underwent gastroscopy with H. pylori isolation and serum testing. Patient diagnostic subgroups were prospectively defined. Genotypes were determined by amplification using polymerase chain reaction. CagA antibodies were determined by western blot, enzyme-linked immunosorbent, and flow microsphere immunofluorescent assays. RESULTS: Patients were grouped as follows: nonulcer dyspepsia (26%), GERD (20%), gastric ulcer (17%), duodenal ulcer (12%), gastric cancer (6%), or controls (19%). The cagA gene was present in 94-97% of subjects in all categories, but the cagA antibody was less prevalent in nonulcer dyspepsia (69%, 95% CI: 48-86%, p = 0.02) and GERD (69%, CI: 39-91%, p < 0.05) than in those with gastroduodenal pathology including gastric ulcer, duodenal ulcer, and gastric cancer (92%, CI: 81-98%). The cagE gene and vacA S1 genotype were more frequent in patients with gastroduodenal pathology (p < 0.01). GERD was associated with a significantly lower rate of vacA S1 genotype than controls (29% (CI: 10-56%) versus 80% (CI: 59-93%), p < 0.01). The vacA S1 genotype was associated with the presence of cagA antibodies. CONCLUSIONS: The cagE and vacA S1 genotypes are more prevalent in patients with peptic ulcer or gastric cancer, suggesting a potential function in virulence for these genes. However, the vacA S1 genotype was also more prevalent in controls than GERD, suggesting a potential protective effect against GERD.

An educational tool to illustrate cost-effectiveness in diagnostic pathways for coronary artery disease.

Rapid increases in healthcare costs have led to increased interest in the cost-effectiveness of medical interventions. Coronary artery disease is responsible for a significant share of total healthcare spending, and therefore economic evaluations of medical procedures to treat the condition are potentially very important. We have developed a spreadsheet model as an educational tool that can be used to illustrate cost-effectiveness in the selection of diagnostic pathways (a "work-up" strategy of tests designed to reach a final diagnosis) for coronary artery disease. The model, in Microsoft Excel, is easy to use, requiring no specialist computer knowledge. It is menu-driven and the user navigates the model via a number of on-screen buttons. A data entry screen allows the user to customize the data for the key model parameters, making it possible to take into account location-specific features. The data entry screen also allows the user to undertake sensitivity analysis and rate "what if" scenarios. The model demonstrates how sensitive the cost-effectiveness of different diagnostic pathways is to the pretest probability of disease. This package could also be used as a decision support tool, although it is important to recognize some of its limitations for this purpose.

An experience of utilization review in Europe: sequel to a BIOMED project.

OBJECTIVE: To develop and test a utilization review screening tool for use in European hospitals. SETTING: In 1993 a group of researchers financed by a European Union grant reviewed the use of utilization review in Europe. They quickly noticed a lack of specifically designed instruments able to take into account the health care and cultural differences across Europe, and available for use in different health care systems. Hence, they embarked upon the task of developing and testing a utilization review screening tool for use in European hospitals. RESULTS: The European Union-Appropriateness Evaluation Protocol's list of reasons was developed and assessed. This is a common taxonomy that classifies days identified as unnecessary and provides a list of levels of care to identify patients' needs. This new protocol not only substitutes for the multiple previous local versions of the Appropriateness Evaluation Protocol, but will also facilitate comparisons of the varying experiences in European countries. MAIN FINDINGS: Development of utilization review in Europe has been carried out mostly on a voluntary basis and the main objective was not control. The experience varies widely: from France, where utilization review is still developing and research has been implemented by local teams, to Portugal, where utilization review programmes have been initiated by government authorities. At this point different initiatives in quality improvement, and more specifically in utilization review, are being developed within the European context.

A European version of the Appropriateness Evaluation Protocol. Goals and presentation. The BIOMED I Group on Appropriateness of Hospital Use.

This paper describes the development and testing of a European version of the Appropriateness Evaluation Protocol (AEP). It stemmed from the original U.S. version and the multiple adaptations and modifications made previously and separately by researchers in European countries. The group was particularly concerned with developing a common list of reasons for inappropriate admissions and days of stay, since the principal goal was to enable an understanding of inappropriate hospital use and potential solutions within local health and social care systems. Developing a common EU-AEP included several steps. First, each national instrument was translated from the national language to English. These back translations were compared with each other and with the US-AEP. A working group analyzed the content of the lists of reasons published in the literature and proposed a novel conceptual approach. On the basis of workshop discussions, a draft of a common European version was circulated to each participant for agreement. In the EU-AEP, the clinical criteria for the appropriateness of admission include 10 related to patient condition and five to clinical services. The criteria for the appropriateness of days of care include 10 covering medical services, six for life support/nursing services, and eight related to patient condition. The proposed core list of reasons of inappropriateness distinguish clearly between two concepts: a) the level of care required by the patient; and b) the reason why this level of care was not used. The first list would thus refer to the nature of resources and facilities required, while the second would focus more on the efficient organization of those resources. A validated European tool to assess inappropriate hospital admissions and hospital days of stay and their causes might be used to assess the need for resources for inpatient care as well as for outpatient care. Assessing the reasons for inadequacies might lead also to the examination of organizational questions. Finally, a common tool allows comparisons between countries concerning the frequency of inappropriate admissions and days of stay and their reasons in relation to the different organizations of health care across Europe.

Economic consequences of early inpatient discharge to community-based rehabilitation for stroke in an inner-London teaching hospital.

BACKGROUND AND PURPOSE: In an inner-London teaching hospital, a randomized trial of "conventional" care versus early discharge to community-based therapy found no significant differences in clinical outcomes between patient groups. This report examines the economic consequences of the alternative strategies. METHODS: One hundred sixty-seven patients received the early discharge package, and 164 received conventional care. Patient utilization of health and social services was recorded over a 12-month period, and cost was determined using data from provider departments and other published sources. RESULTS: Inpatient stay after randomization was 12 days (intervention group) versus 18 days (controls) (P=0.0001). Average units of therapy per patient were as follows: physiotherapy, 22.4 (early discharge) versus 15.0 (conventional) (P=0.0006); occupational therapy, 29.0 versus 23.8 (P=0.002); speech therapy, 13. 7 versus 5.8 (P=0.0001). The early discharge group had more annual hospital physician contacts (P=0.015) and general practitioner clinic visits (P=0.019) but fewer incidences of day hospital attendance (P=0.04). Other differences in utilization were nonsignificant. Average annual costs per patient were pound sterling 6800 (early discharge) and pound sterling 7432 (conventional). The early discharge group had lower inpatient costs per patient (pound sterling 4862 [71% of total cost] versus pound sterling 6343 [85%] for controls) but higher non-inpatient costs (pound sterling 1938 [29%] versus pound sterling 1089 [15%]). Further analysis demonstrated that early discharge is unlikely to lead to financial savings; its main benefit is to release capacity for an expansion in stroke caseload. CONCLUSIONS: Overall results of this trial indicate that early discharge to community rehabilitation for stroke is cost-effective. It may provide a means of addressing the predicted increase in need for stroke care within existing hospital capacity.

Variations in case fatality and dependency from stroke in western and central Europe. The European BIOMED Study of Stroke Care Group.

BACKGROUND AND PURPOSE: There are significant variations in mortality rates from stroke in Europe. A European Union BIOMED Concerted Action was established to assess and determine the reasons for the variations in case fatality and disability after stroke. METHODS: Hospital-based stroke registers were established in 12 centers in 7 western and central European countries to collect demographic, clinical, and resource use details at the time of first-ever stroke during 1993-1994. At 3 months, details of survival, activity of daily living score, and use of health services were recorded. Multinomial logistic regression was used to estimate the relationship between centers and outcome (dead, functionally independent, functionally dependent), with adjustment for case mix and resource use variables, and to predict outcomes for the full cohort. This should minimize the bias due to loss to follow-up. RESULTS: A total of 4534 stroke events were registered. The mean age was 71.9 years (SD, 12.53). There were significant differences between centers for all case mix and resource use variables (P<0. 001). Multinomial logistic regression modeling of outcome indicated that for those patients initially unconscious (588), center was not significantly related to outcome (P=0.427). For those initially conscious, there were wide variations in death and dependency between centers after adjustment for case mix, type of bed, and use of CT scan. The predicted proportion dead at 3 months ranged from 42% (95% CI, 35% to 49%) in one UK center to 19% (95% CI, 14% to 24%) in France. CONCLUSIONS: Areas with high mortality rates within western and central Europe have been identified for stroke outcome, and there appears to be opportunity for considerable health gain in certain centers. Adjustment for case mix and health service resource use does not explain these differences in outcome. Although there are true differences in outcome, the aspects of care that need to be altered to improve outcome remain unclear despite detailed data collection. Comparisons of outcome of the same design used in the present study do not allow rational policy decisions to be made.

Cloning, sequencing, and developmental expression levels of a novel glutamate-gated chloride channel homologue in the parasitic nematode Haemonchus contortus.

Glutamate-gated chloride channels (GluCls) are the proposed site of action for macrocyclic lactone anthelminthics such as ivermectin (IVM) and the milbemycins such as moxidectin (MOX). The importance of this interaction between macrocyclic lactones and GluCls is strengthened by the recent genetic evidence that GluCls are involved in IVM and MOX resistance in the parasitic nematode Haemonchus contortus (1). We have cloned two full length GluCl putative alpha-subunit cDNAs from H. contortus (HcGluCla and b) that exhibit different sized ligand binding domains. Phylogenetic analysis of the conserved regions of the amino acid sequence of HcGluCla suggests that it is a member of the GluCl family but forms a distinct subbranch within this family. The expression level of HcGluCla was examined in different developmental stages of H. contortus (eggs, L3, and adults) and found to be significantly downregulated in eggs compared to adults.