This method has been shown to produce reliable results on simulated data and on human data from the TAP2 region. Strikingly, however, it finds very little support for recombination rate variation at TAP2 in the western chimpanzee data. Moreover, simulations suggest that there should be stronger support if there were a hotspot similar to the one characterized in humans. Thus, it appears that the human TAP2 recombination hotspot is not shared by western chimpanzees. These findings demonstrate that fine-scale recombination rates can change between very closely related species and raise the possibility that rates differ among human populations, with important implications for linkage-disequilibrium based association studies.Recent experiments using sperm typing have demonstrated that, in several regions of the human genome, recombination does not occur uniformly but instead is concentrated in “hotspots” of 1–2 kb. Moreover, the crossover asymmetry observed in a subset of these has led to the suggestion that hotspots may be short-lived on an evolutionary time scale. To test this possibility, we focused on a region known to contain a recombination hotspot in humans, TAP2, and asked whether chimpanzees, the closest living evolutionary relatives of humans, harbor a hotspot in a similar location. Specifically, we used a new statistical approach to estimate recombination rate variation from patterns of linkage disequilibrium in a sample of 24 western chimpanzees  The human TAP2 recombination hotspot is absent from the homologous region in western chimpanzees, with important implications for association studies, the HapMap project and understanding fine-scale variation in recombination rates Understanding recombination is also of practical importance for evolutionary inference and human genetics . UnfortuOne way to learn about finer-scale recombination rates in males is sperm typing . In thisD. melanogaster and D. simulans, two sibling species that differ in their recombination landscape , another Old World monkey. For more closely related species, nothing is known. However, observations in yeast . The two species are thought to have had a common ancestor five to six million years ago and differ at approximately 1.2% of base pairs on average  but not western (P. t. verus) chimpanzees , consistent with previous findings  and Pr(λ > 10); these are the posterior probabilities of a hotspot of any kind and of a hotspot of intensity at least ten times the background rate, respectively.The LD data are summarized in t, λ see . Within sian see  so, as aλ > 1) = 1 and Pr(λ > 10) = 0.982. When the same method was applied to the human genotype data , we estimated Pr(λ > 1) = 1 and Pr(λ > 10) = 0.992. The results are almost identical, suggesting minimal loss of information with the use of genotypes. Interestingly, the point estimate of λ using either haplotypes (28.4) or genotypes (32.1) is higher than the corresponding estimate from sperm typing (11). This difference may reflect error in the estimates; alternatively, it may point to a more intense hotspot in females than in males  = 0.200 and Pr(λ > 10) = 0.006, reflecting tepid support for a hotspot of any kind and almost no support for a hotspot similar to the one observed in humans. Indeed, the latter figure represents very strong evidence against a hotspot of moderate intensity and rules out the possibility that the chimpanzee polymorphism data are simply uninformative, because of, for example, insufficient sample size or diversity.Next, we applied the same method to the genotype data collected from western chimpanzees. The estimate of the background rate of recombination, ρ^ype data . Howeverλ = 11 and then tabulated the proportion with posterior probability estimates as low or lower than that observed , the probability of obtaining Pr(λ > 1) ≤ 0.200 is p = 0.010 and the probability of obtaining Pr(λ > 10) ≤ 0.006 is p = 0.005. With a lower ρ value  ≤ 0.200 is p = 0.020. In other words, we can reject the null hypothesis that there is a hotspot in western chimpanzees similar to the one in humans, because we would expect to see more support for a hotspot in these data if one were there. It appears that western chimpanzees do not harbor a hotspot in the same location as humans.To assess how likely we would be to obtain such weak support if there were in fact a hotspot in western chimpanzees similar to the one in humans, we generated 200 simulated genotype data sets under a model with a hotspot of intensity rved see . We tookρ and λ . This in turn raises a number of more general questions. Are hotspots frequently born de novo or do they tend to migrate within circumscribed regions of the genome? Are particular sequence motifs sufficient to produce recombination hotspots, or are larger-scale requirements, such as chromatin accessibility, required for their formation ? The sysComparative studies of hotspot regions will also increase our understanding of the determinants of mutation rates. As noted by In conclusion, these analyses demonstrate that fine-scale recombination rates can change between closely related species. Together with the observations that crossover frequencies can depend on specific haplotypes  and thatThe extent to which local recombination rates vary among human populations influences the degree of similarity of LD patterns among them, with important consequences for the design of efficient LD-based association studies  that were wild caught or known to be unrelated based on recent pedigrees. Twelve samples  are from the collection stored at the Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany, while 12 other samples  were kindly provided by P. Morin and the Primate Foundation of Arizona.We used DNA from 24 western chimpanzees We amplified 9,491 bp from the TAP2 region, corresponding to base pairs 113102–122585 of the sequence from 2, PCR buffer , 0.5 U of Taq DNA polymerase , and 10 pmol of each primer. We used 50–100 ng of DNA in each 30 μl PCR. Amplification conditions for all regions were the following: incubation for 3 min at 94 °C, 35 cycles  and a final elongation of 5 min at 72 °C. A nested PCR was performed to obtain regions 6 and 7 by using the product of the primers Tap2 5 5′ and Tap2 8 3′ as a template. PCR products were separated from primers and unincorporated dNTPs by treatment with a solution of 10% PEG 8000/1.25 M NaCl followed by centrifugation. PCR products were then air dried and resuspended in 10–15 μl of H2O.DNA amplification reactions contained 250 μM of each dNTP, 1–2 mM MgClSequencing reactions consisted of 1 μl of ABI Prism BigDyeTM Terminators version 2.0 , 8–10 ng of purified PCR product, and 1 μl of 2.5 μM primer (the same primers used for PCR) in a volume of 7 μl. Cycling conditions were 96 °C for 2 min and then 35 cycles of 96 °C (20 s), annealing temperature (30 s), and 60 °C (4 min). Isopropanol-precipitated cycle sequencing products were run on an ABI 3730 DNA analyzer. Base calling was done with ABI Prism DNA Sequencing Analysis version 5.0 and ABI Basecaller. BioEdit version 5.0.6 was used for sequence analysis and alignment.In total, 2-fold coverage of a 9,370 bp sequence was obtained for each individual; these are available from GenBank see . Most ofθ = 4Neμ (Ne is the diploid effective population size and μ is the mutation rate per generation), using Watterson's estimator, θW  to allele frequencies in western chimpanzees. Specifically, we ran 104 coalescent simulations of the standard neutral model with the same number of chromosomes and base pairs as in the actual data, with θ equal to θW, and with the population recombination rate equal to the estimated value (see below). We then tabulated the proportion of simulated runs with a Tajima's D value as or more extreme than that observed.We estimated the population mutation rate, tor, θW , based o, θW  model of H given the underlying recombination parameters α . This can be used directly to estimate α from H in situations where haplotypes have been experimentally determined . To do so, we start with an initial guess for the haplotypes, and iterate the following steps: (i) estimate a new value for α, using the current estimate of H and (ii) estimate a new value for H, using the genotypes G and the current value for α. Step (i) is performed using the PAC-B model of α described below. Step (ii) is performed by using the method for haplotype inference described in i = ziρ/(j+ ziρ)q in their Appendix A with i =1−exp(−ziρ/j)q.) Both the PAC-B model and the Fearnhead and Donnelly conditional require the specification of a mutation parameter, θ, and a mutation process. In each case, we used the value of θ given in More formally, the model of ed e.g., . HoweverH, α | G) from which we wish to sample. Provided that the algorithm is run for sufficiently long, the estimates of α obtained each iteration provide a sample from the distribution Pr(α | G), and thus allow α  to be estimated directly from G, taking full account of the fact that the actual underlying haplotypes are not known. The algorithm is implemented within the software package PHASE version 2.1, which is available online at http://www.stat.washington.edu/stephens/software.html.This iterative scheme defines a Markov chain whose stationary distribution is the distribution Pr of constant rate r (per base pair) outside the hotspot and of constant rate λr inside the hotspot; gene conversion is not explicitly modeled. In the first version, we assumed that, if present, the hotspot is at the same location as estimated by sperm typing in humans (4180–5417).  and λ. We assumed a priori that a hotspot exists with probability 0.5 and that, if the hotspot exists, λ is between one and 100. Specifically, we assumed that λ = 1 with probability 0.5 and otherwise that log10(λ) is uniformly distributed on . The prior on ρ is uniform on log10(ρ) in the range , which covers all plausible values.We considered two versions of the simple hotspot model of λ and ρ. In this case, we assumed a priori that the hotspot exists with probability 0.18 (corresponding to an assumption that a hotspot occurs roughly once per 50 kb of sequence), that the center of the hotspot is equally likely to be anywhere along the length of the sequence, and that the width of the hotspot is between 200 and ∼4,000 bp . Priors on ρ and on λ  are as in the first version.In the second version, we assumed that the location and width of the hotspot are unknown and to be estimated along with To allow for potential problems with convergence of this Markov chain Monte Carlo algorithm, we ran the algorithm ten times for each analysis, using different seeds for the pseudorandom number generator. For each run, we obtained a point estimate of the parameters (using sample posterior medians) and posterior probabilities. The reported estimates are the median of the estimates obtained from the ten runs.λ = 11, and θ = θW. Haplotypes were randomly paired to form genotypes and phase information was ignored. The data were masked to mimic the actual data structure, i.e., they included a gap of 487 bp in the same position. We then counted the proportion of simulated data sets for which our estimate of the posterior probability was as low as observed or lower .Click here for additional data file.http://www.ncbi.nlm.nih.gov/) accession number for the human TAP2 region of The GenBank (


Atrial fibrillation (AF) has been recognised as an important independent risk factor for thromboembolic disease, particularly stroke for which it provides a five-fold increase in risk. This study aimed to determine the baseline prevalence and the incidence of AF based on a variety of screening strategies and in doing so to evaluate the incremental cost-effectiveness of different screening strategies, including targeted or whole population screening, compared with routine clinical practice, for detection of AF in people aged 65 and over. The value of clinical assessment and echocardiography as additional methods of risk stratification for thromboembolic disease in patients with AF were also evaluated.The study design was a multi-centre randomised controlled trial with a study population of patients aged 65 and over from 50 General Practices in the West Midlands. These purposefully selected general practices were randomly allocated to 25 intervention practices and 25 control practices. GPs and practice nurses within the intervention practices received education on the importance of AF detection and ECG interpretation. Patients in the intervention practices were randomly allocated to systematic (n = 5000) or opportunistic screening (n = 5000). Prospective identification of pre-existing risk factors for AF within the screened population enabled comparison between high risk targeted screening and total population screening. AF detection rates in systematically screened and opportunistically screened populations in the intervention practices were compared to AF detection rate in 5,000 patients in the control practices. Atrial fibrillation (AF) has been recognised as an important independent risk factor for thromboembolic disease, particularly stroke with which it is associated with a five fold increase in risk . There aScreening for AF in the elderly fulfils many of the Wilson-Jungner criteria for a screening programme . It is aOne UK study has compared systematic nurse-led screening with prompted opportunistic case finding for AF in primary care . This smopportunistic case finding, where a health care professional took the opportunity to feel a patient's pulse during a consultation. If the pulse is irregular, they might make a clinical diagnosis of AF, or request/perform an electrocardiogram (ECG) as a confirmatory test. However, opportunistic case finding is likely to miss a significant proportion of people who would otherwise have benefited from treatment. For example, detection of hypertension in general practice was traditionally detected in an opportunistic way until the introduction of health checks with the 1990 GP contract. The Health Survey for England shows that in 1991, 42% of the population over the age of 75 had hypertension for which they were not taking any medication [The simplest strategy was dication . This fitargeted screening programme. Cardiac failure, hypertension and rheumatic heart disease are important precursors of AF [One possible approach was to screen patients who are at higher risk of AF – a rs of AF . AF is mrs of AF . Most gewhole population screening programme.Another approach was to screen everyone 65 and over (65+) for AF – a A modelling exercise using decision analysis to inform on the methodology for this study indicated that there were not sufficient primary data available to recommend which of these (targeted or whole population) would be the optimum policy12-lead ECG is recognised as the gold standard test, but this test is time consuming (taking at least 15 minutes to perform in an outpatient setting). Therefore, it is important to consider simpler tests. This study assessed simpler methods compared to the gold standard, both in terms of accuracy, time taken and patient acceptability. These include taking the pulse, and simpler ECGs.Cardiologists offer the most accurate readings of ECGs, but can satisfactory interpretations be obtained by the GP, the practice nurse, or computerised diagnostic software? This study assessed the accuracy of these different approaches to interpreting the ECG.The main treatment options to reduce risk of stroke in patients' with AF are currently warfarin or aspirin. Aspirin is much less effective than warfarin – it achieves a barely significant 21% reduction in stroke risk . HoweverThis study, by providing answers to these questions, allowed the optimum strategy for introducing a screening programme for AF in the over 65s to be determined. However, before a decision is made as to whether to institute a screening programme, not only must the question of the best strategy be considered, but also, the question of whether any screening programme at all should be introduced. This study provided data to assist in answering this fundamental question by providing:i) An accurate estimate of the community prevalence and incidence of AF in over 65s;ii) An assessment of the health economic implications of screening for AF;iii) An assessment of the service provision implications of implementing such a programme;iv) An assessment of the impact on patient quality of life and anxiety after various screening methodologies.Although the cost effectiveness of different approaches to screening is often put in terms of the average cost per case detected, such an approach ignores the sensitivity and specificity of the screening test. This is because average cost per case detected focuses entirely on true positives, paying no attention to false positives, false negatives and true negatives. False positives and false negatives impose costs on patients and health services which would be neglected if the focus was confined to true positives . An unduThis study compared the incremental cost per case detected for different methods of AF screening. This refers not to the average cost but rather approximates the incremental cost per case detected in moving from one of the screening options to another. Use of incremental cost per case detected by option shows how the cost per additional case detected is likely to increase as the intensity of screening increases. This method has been used to deal with similar uncertainties about the cost effectiveness of screening for other diseases, including breast and colorectal cancer and has been recommended by the US guidelines .• To determine baseline prevalence and the incidence of AF based on a variety of screening strategies and in doing so to evaluate the incremental cost-effectiveness, in terms of cost per case identified, of the different screening strategies (targeted or whole population screening) compared with routine clinical practice for detection of AF in people aged 65 and over.• To evaluate the relative cost-effectiveness of screening methods for AF diagnosis, comparing 12 lead ECG (gold standard) with pulse taking, lead II rhythm strip from standard ECG limb leads alone and single lead thoracic placement ECG.• To evaluate the most cost-effective method of test interpretation, comparing cardiologist (gold standard), with GP, practice nurse, or computerised diagnostic software.• To assess the differing combinations of screening strategies and procedures in terms of patient acceptability and impact on patient quality of life, including any psychological effects of screening.• To determine the community prevalence of AF in people 65+.• To evaluate the value of clinical assessment and echocardiography as additional methods of risk stratification for thromboembolic disease in patients with AF.• To evaluate the service provision implications should screening for AF become a national programme, and identify the optimum screening algorithm for identification of patients with AF.• The incidence of AF according to a variety of screening strategies• The associated costs providing an incremental cost per case detected. The cost data was collected from an NHS and patient perspective. It has focused on resources required to establish screening, time taken to complete screening and the cost of the equipment.• Cost effectiveness of 4 different methods of screening for AF. The cost data focused on the difference in the cost of the equipment and the time taken for each of the different methods of screening to be completed. This was from both an NHS and patient perspective.• Cost effectiveness of 4 different methods of ECG interpretation. The cost data focused on the difference in the cost of the grade of staff interpreting the ECG and the accuracy of their interpretation.• Overall community prevalence and incidence of AF• Patient acceptability to AF screening was measured using an adapted version of the screening specific questionnaire used in the Colorectal Screening Programme . Patient• Modelling techniques were used to identify the implications of AF screening on health service provision nationally. This included the effect on echocardiography and anti-coagulation clinic provision.This was a multi-centre randomised controlled trial. The study schema is shown in figure 50 computerised general practices within the West Midlands were recruited through MidReC (Midlands Research Practices Consortium). This was undertaken by writing to all practices in the West Midlands and surrounding counties explaining the study and asking whether they were interested in participating. Practices showing an interest were given further information about the study and invited to attend an investigators meeting. Following the investigator meetings sixty practices interested in participating in the project were randomised (stratified based on Townsend score and practice list size): 25 as intervention, 25 as control practices with 10 reserve practices.1/3rd of the total population of patients 65+ in this group), and 5000 from the control practices  were identified.A computerised list of all patients aged 65+ was obtained from each practice, and from this a random sample of 10,000 patients from the intervention practices , radial pulse and a 12 lead ECG. The 12 lead ECG was performed using an electronic ECG machine which allowed print-out of single lead thoracic placement ECG and a rhythm strip of lead 2 using limb leads from standard ECG.All 12 lead ECGs were sent to two cardiologists for reporting . Where there was disagreement over the diagnosis a third cardiologist was used to decide. The cardiologists were asked to state whether the ECG showed AF or not, and to state whether there were any other significant abnormalities. Patients were informed of the result within two weeks. Patients with normal ECGs were informed of this, patients with any abnormality were asked to make an appointment with their GP. At the GP appointment patients with AF aged 65 – 74 were offered echocardiography. GPs were asked to make a clinical decision as to thromboprophylaxis both before and after the echocardiogram. Patients with other ECG abnormalities were managed as clinically indicated.At the end of the screening process, GPs and Practice Nurses from both intervention practices (who had received education on ECG interpretation) and control practices (who had received no education) were sent ECGs to interpret for the presence or absence of AF. All ECGs recorded within the study were printed off as either 12 lead, single lead thoracic placement or limb lead recordings. Allocation to ECG type was random and resulted in three equal ECG groups. In order for each interpreter to read all three types of ECG, batches of ECGs were collated with the same numbers of each type of ECG. Allocation to a batch was also random. In total, there were 25 batches of ECGs to match the number of practices in each arm. The GP and Practice Nurse from the same practice read the same batch of ECGs and each batch was read by one control practice and one intervention practice. Therefore each ECG was read by two GPs and two Practice Nurses. All ECGs were anonymised, and practices did not receive any ECGs from their own practice. The interpreters were given a sheet to fill in to indicate for each ECG the presence or absence of AF. All ECGs (as 12 lead) were also analysed by the specific software package accompanying the electronic ECG and results recorded.Patient acceptability and quality of life for different screening strategies were established using EuroQol (EQ-5D) combined together with the Speilberger 6-item Anxiety Questionnaire. EQ-5D allowed the measurement of broad aspects of quality of life. The shortened Speilberger anxiety questionnaire also has proven validity and is more specific to anxiety than is the SF-12 . An adapA random sample of 750 patients  were sent postal versions of the psychological instruments (EQ-5D and Spielberger) on entry to the study (i.e. before the intervention group has received their invitation to attend for screening). One reminder was sent a month later to non-responders. The same questionnaires were sent to the same groups plus those patients who had screened postive at the end of the screening period, approximately 17 months later. This allowed a non-randomised comparison between the effects on quality of life and anxiety in screen positive and screen negative patients.In addition, all patients who were screened were asked to complete the acceptability and Spielberger questionnaire immediately after screening. The patient acceptability questionnaire was also administered to all patients who proceeded for echo.The value of clinical assessment and echocardiography in risk stratification were determined in patients aged 65–74. This compared GP assessment based on the Birmingham guidelines for thromboprophylaxis in AF with any changes in recommendations for treatment once echocardiography results were available to the GP.The assumptions for the power calculations were that patients aged 65 and over represent 17% of the total population; that 40% of study population will be in the high risk group.Also assuming that:1. Minimum worthwhile change in detection rate was 1% for targeted screening versus routine practice. It is estimated that this change would equate to £10,000 per life year gained. This is based on the following assumptions:a) 60% of new cases of identified AF would be suitable candidates for warfarinb) Annual risk of stroke in this population was 5%, reduced by 60% to 2% if treatedc) Costs: £25 to screen a patient; £100 to treat with warfarin pa; £6,000 NHS costs to treat a stroke2. 50% of patients with AF will be already known to their general practitioner (estimates range from 30%  to 76% )3. Community prevalence of AF in this population was 6% See figure All sample size calculations were for 90% power and 5% significance levels unless otherwise stated.a) To detect a 1% difference in detection rate between intervention (GP educated) and control practices (B2-A1) vs (C2-C1). This requires 1,236 patients. However, since this is a difference based at the practice level of randomisation, it needed to be inflated by the design factor. Based on AF prevalence data from the EcHoES (Echocardiographic Heart of England Screening) Study [actices B-A1 vs  and control practices (A3-A1) vs (C2-C1)b) To detect a 1% difference in detection rate between intervention  and control practices (A2-A1) vs (C2-C1)c) To detect a 1.8% difference in detection rate between intervention  required fewer patients to detect the expected difference (2%) stated in the assumptions, it would be possible to detect differences as low as 1%, should the detection rate not be as high as expected.The a), b) and c) comparisons are all at practice level randomisation.d) To detect a 1% difference in detection rate between high risk screening strategy and routine practice prompted by education (opportunistic arm) (A2-A1) vs (B2-A1).This requires 1,236 patients in both the high risk systematic screening and the GP educated (opportunistic) screening arms of the intervention practices assuming the high risk screening detects a 1% increase and opportunistic screening detects 0% increase. Should the increased detection rates be higher in each arm (1.7% in the high risk arm and 0.7% in the opportunistic arm) then this could require 2,686 patients in each arm. However, since there is a ratio of 2:5 patients in these arms there will be sufficient patients as only 1,880 are needed in the high risk arm and 4,700 in the opportunistic arm to be able to detect this 1% difference.e) To detect a 1% difference in detection rate between total screening strategy and routine practice prompted by education (opportunistic arm) (A3-A1) vs (B2-A1).This requires 3,300 patients in both the total screening and the GP educated (opportunistic) screening arms of the intervention practices.f) To detect a relative risk (RR) of 2 (1% detection rate difference) between total population and high risk screening (A3 vs A2).It was assumed that 40% of the study population fall into the high risk group, and the prevalence of undetected AF is 3%. This meant that 1,434 patients would be needed in each of the two risk groups to detect a two fold difference in risk (i.e. RR of AF in high risk as compared to low risk group is 2). This RR of 2 equates to an increase in AF detection rate from 3% in the total population arm to 4% in the high risk arm. Since there was a 40:60 split in the two risk groups unequal sample size calculations only require a minimum of 1,200 patients in the high risk group and 1,800 in the moderate/low risk group. This was achievable with a screening arm of 5,000 patients, as there would actually be 1,320 in the high risk group and 1,980 in the moderate risk group if a 66% screening acceptance rate was assumed.Although some of the variances are from North American populations we have no reason to suspect that the variation will be different in a British population since data from the ECHOES study on the SF36 gives variations very similar to the North American norms.The shortened (6-item) version of the Spielberger state anxiety questionnaire has been validated and used in populations different from that under consideration in SAFE, namely it tends to have been used in young and mostly female populations ,21,22. TThe VAS variance as reported for an elderly population aged 75 and over was 365  but for This was reported in different ways in the Johnson  and DormIntention to treat analysis will be used.Any previously known Atrial Fibrillation cases will be subtracted from the totals obtained at the end of the study to ensure there is no double counting in the incidence figures.Chi-squared, independent t-tests and log-linear models will be used to describe demographic data. If there are differences between the groups this may need to be adjusted for in later analyses.Proportions and rates will be used as the measures of prevalence and incidence.The independent t-test and ANOVA with random effects (as appropriate) will be used to examine the detection rate differences between the intervention and control screening strategies. Should the data be strongly non-normal a non-parametric equivalent will be used.a) to assess patient acceptability and impact on QoL of different screening strategiesThe independent and related t-test and ANOVA will be used to examine the differences between the intervention screening strategies on the Spielberger and EuroQol EQ5D. Should the data be strongly non-normal a non-parametric equivalent will be used. Chi squared tests will be used on the screening tool.b) to assess the value of echocardiography in risk stratification for thromboembolic disease in patients with AFMcNemar's test will be used to see whether there is any significant change in the doctor opinion on risk of CVA and treatment decision before and after echo screening.c) to evaluate the most cost effective method of test interpretationThe cost effectiveness will be covered in the economic section. However, the use of sensitivity, specificity, Cohen's κ and conditional logistic modelling will allow for comparison of the various methods for detecting AF between the GPs, nurses and consultants.d) to evaluate the most cost effective method of screeningThis will be covered in the economic analysis section.Multivariate and logistic modelling analyses will be undertaken in order to determine which markers might be the best predictors of the presence of AF. This will act as confirmatory analysis for the risk factors used in the screening strategy to define a high risk patient.This trial evaluated a large number of alternative screening scenarios for identifying atrial fibrillation . The study has been powered to detect a difference in the targeted versus population arms, and in systematic screening versus routine clinical practice. However, the economic analysis will compare the cost-effectiveness of all alternative screening approaches using a modelling framework, whereby data will be drawn from the trial (where appropriate and available) and from external sources.The use of a modelling approach allows the timescale for the economic analysis to be extended beyond the follow-up period allowed for in the trial. The use of such extrapolation will enable estimation of the incremental cost-effectiveness ratios (ICERs) for each approach:Incremental cost per case detectedIncremental cost per life year gainedIncremental cost per quality-adjusted life year (QALY) gainedThe use of EQ-5D allows the measurement of broad aspects of quality of life. EQ-5D allows changes in health status to be measured but also valued, using the University of York Measurement & Valuation of Health general population survey tariff .The cost analysis adopted a broad perspective to include costs incurred within the health sector and by patients and carers. Data collection was undertaken on all trial patients in order to allow a stochastic cost analysis to be conducted. The focus of the data collection will be upon the key cost drivers which will include:a) the resources required to establish screening ,b) the time taken to carry out the various tests, andc) the cost of the equipment (expressed as cost per test).a), b) and c) will be based on data collected in the study.The analysis adopted an incremental approach such that data collection concentrated on resource use differences between alternative screening scenarios. The process of collecting data on resource use was undertaken separately from data collection on unit costs. Resource use data on the screening process was principally collected within the trial. Unit costs were collected from published sources and a representative sample of NHS providers in order to increase generalisability. The methods used in collecting data will include patient questionnaires (see above) and review of patient records . Data on private costs were collected from a survey of a sub-cohort of the trial population.The plan for the analysis is:1. Report a cost consequence analysis, which will involve providing a full description of all important results relating to costs and consequences.2. Conduct both a cost-effectiveness analysis and a cost utility analysis (using data on true positive cases detected as the measure of effect and data on EQ-5D to estimate QALYs).An incremental approach will be used in order to compare the large number of alternative screening strategies.We are interested in comparing the mean costs per patient since our concern is with predicting overall programme costs. However, the data on costs are likely to have a skewed distribution. Therefore, the plan for the analysis of costs is:1. To explore the nature of the distribution of costs2. If required, to use non-parametric comparison of means (e.g. bootstrapping)3. If the distribution of the data is approximately normal, parametric methods will be used.This approach is in line with recent recommendations .If missing data are a problem at the economic analysis stage, then imputation techniques will be employed.Longer term costs and consequences will be explored by extrapolating beyond the end of the trial using a modelling framework using data from a range of trial and non-trial sources. The precise form of modelling is yet to be determined, but is likely to be either Markov or Discrete Event Simulation, depending upon the extent to which the Markov assumptions are justified. An advantage of using such an approach is that it will allow the additional costs of increasing survival to be explicitly incorporated into the analysis. In particular, modelling will provide estimates of the optimal frequency of screening for AF, based on the estimates of incidence and prevalence from the trial.Identifying untreated patients with AF will have implications for service provision. These will depend upon the prevalence of atrial fibrillation, the screening mechanism employed, the use made of echocardiography, and the additional requirements for anticoagulation monitoring. The separate parallel study, BAFTA, will provide empirical data that will allow such implications for service provision to be assessed. The modelling exercise, which will draw on both SAFE and later BAFTA results, will combine best estimates of both screening and anticoagulation options.The robustness of the results of the economic analysis will be explored using sensitivity analysis . This wiPatients aged 65 years or over (65+).Patients who were terminally ill.Randomisation of practices and patients was performed by statisticians from the Department of Primary Care and General Practice at The University of Birmingham. Cluster randomisation of practices to intervention or control was stratified by Townsend quartiles and practice size. Computer searches were carried out to identify cases of known AF, within the sample of patients identified above, using a published strategy . The ranFollowing initial sampling of the total population the list of patients from each practice were returned to the practices who were asked to remove any patients who had died, moved or were terminally ill. Patients removed following this process were replaced with patients from a reserve list, which had been randomised at the same time as those on the initial lists.Patients aged 65+ who were selected to the systematic screening arm received an information sheet with an invitation letter to attend the ECG clinic. Entry to the trial was discussed with the practice nurse at the clinic. The practice nurse then obtained written consent from those patients who were willing to participate.Study patients found opportunistically to have an irregular pulse were given an information sheet and invited to attend the screening clinic.GPs and other members of the primary health care team in the intervention practices attended investigator days at which they were given educational materials informing them of the importance of detection of AF, and the treatment options that are available. The materials encouraged them to consider opportunistic screening of patients.Members of the primary health care team in control practices received no educational input from the research staff.Practice nurses attended an ECG training day prior to starting the ECG screening clinics. Training included how to perform an ECG (using the Biolog) to ensure a standardised high quality tracing and basic ECG interpretation .Computer searches were carried out to identify cases of probable AF in the 15,000 study patients using a published strategy [Case notes of patients identified as 'known' or 'probable' AF in any of these computer searches were reviewed for mention of a diagnosis of AF. AF diagnosis were drawn from hospital letters stating the existence of the condition or ECG recordings from the last 5 years.An additional 5% random sample of case notes of patients not identified as 'known' or 'probable' AF by computer searching were reviewed  to estimate how many other patients who are known to have AF were not identified by the computer searches. If this had revealed a significant number of extra cases of known AF, then the sample size for manual searching would have been increased to allow a precise estimate of the baseline rate of known AF. Unidentified extra AF cases were not found to be significant so no additional note search was required.The same computer searches on both intervention and control practice patients notes were performed prior to, and 12 months after, commencement of screening.All patients in the systematic screening arm and those found to have an irregular pulse in the opportunistic screening arm of the study were invited to attend an ECG screening clinic. At the clinic the practice nurse explained the aims of the study and answered any questions about the study. Written informed consent to participation in the study was obtained from the patient. The nurse then recorded baseline information on age, sex, present smoking and alcohol status and past medical history, including previous diagnosis of AF, and any treatment the patient may be receiving for AF. Radial pulse rate, and whether regular or irregular, was noted. A 12 lead ECG, the gold standard by which other traces were compared, was then recorded using the Biolog machine, which was also able to produce a trace corresponding to the single lead thoracic placement and a rhythm strip of lead II. Finally, the patient was asked to complete an acceptability questionnaire.This study will identify the most cost-effective strategy for identifying atrial fibrillation in patients aged 65 and over. The policy implications will be dependent on the findings and one of the strengths of the current study is the utilisation of modelling techniques to investigate the implications of different screening strategies and frequency of screening within different health care environments. The initial draft of the report has been submitted to the Department of Health and publication of the results should be expected later this year (2004).None declared.RH and DF were principal investigators. EM and SJ were project managers. DS collected data and prepared the first draft of the paper. JM, EM, JR, SB, MD and GL all contributed to the project design. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Pilot studies are often used to transition therapies developed using animal models to a clinical setting. Frequently, the focus of such trials is on estimating the safety in terms of the occurrence of certain adverse events. With relatively small sample sizes, the probability of observing even relatively common events is low; however, inference on the true underlying event rate is still necessary even when no events of interest are observed. The exact upper limit to the event rate is derived and illustrated graphically. In addition, the simple algebraic expression for the confidence bound is seen to be useful in the context of planning studies. While pilot studies may vary in the fundamental objectives, many are designed to explore the safety profile of a drug or a procedure ,2. Oftenα (the Type I error rate) and β (the Type II error rate) may be inappropriate since the objective of the research is not to provide definitive support for one treatment over another [In the context of pilot studies, traditional levels of  another . For exaβ) is of less practical importance in a single arm, non-comparative  pilot study since the results would almost always require confirmation in a controlled trial setting. Shih et al [Similarly, power % confidence interval is to be generated for π and an estimate of the sample size, n, is desired. Denote X as the number of patients sampled who experience the adverse event of interest. Then, the probability of observing x events in n subjects follows the usual binomial distribution. Namely,For ease of presentation, assume the pilot study will involve πu as the upper limit of the exact one-sided 100 × (1 - α)% confidence interval for the unknown proportion, π [πu is the value such thatDenote rtion, π . Then πux = 0), equation (1) reduces toA special case of the binomial distribution occurs when zero events of interest are observed. In pilot studies with relatively few patients, this is of practical concern and warrants particular attention. When zero events are realized n = α.(1 - α)% confidence interval for π isAccordingly, the upper limit of a one-sided 100 × (1 - πu = 1 - αn1/.     (2)α)% one-sided confidence interval is .The resulting 100 × % confidence that the true rate did not exceed a pre-specified π, say π0, given that zero adverse events were observed. Using (2), it follows that:Furthermore, one can consider using (2) in other clinically important manners. For instance, an investigator may be planning a pilot study and want to know how large it would need to be to infer with 100 ×  declare that they have no competing interests.RC and RW contributed to the conceptualization, writing and editing of this manuscript.


Although significant response time deficits (both reaction time and movement time) have been identified in numerous studies of patients with Parkinson's disease (PD), few attempts have been made to evaluate the use of these measures in screening for PD.Receiver operator characteristic curves were used to identify cutoff scores for a unit-weighted composite of two choice response tasks in a sample of 40 patients and 40 healthy participants. These scores were then cross-validated in an independent sample of 20 patients and 20 healthy participants.p < 0.025) with the motor score of the Unified Parkinson's Disease Rating Scale (UPDRS).The unit-weighted movement time composite demonstrated high sensitivity (90%) and specificity (90%) in the identification of PD. Movement time was also significantly correlated (r = 0.59, Measures of chronometric speed, assessed without the use of biomechanically complex movements, have a potential role in screening for PD. Furthermore, the significant correlation between movement time and UPDRS motor score suggests that movement time may be useful in the quantification of PD severity. Nevertheless, a misdiagnosis of PD can cause psychological trauma and unnecessarily expose patients to PD drugs. Additionally, as new, and possibly neuroprotective, drugs become available for the treatment of PD, early and accurate diagnosis will become increasingly important. As the diagnosis of PD is usually based on subjective clinical assessment of overt symptomatology , the neemography ), but thMontgomery et al. ,4 have pThe global slowing that is consistently demonstrated by PD patients suggests that measures of cognitive or motor speed are logical methods for obtaining quantitative measurements of PD severity. As reaction time (RT) and movement time (MT) have repeatedly been demonstrated to show substantive and significant deficits in PD populations , these The goal of this study, therefore, is to evaluate the diagnostic properties of a choice reaction time task that uses a simple external response console (i.e. a "button box"), similar to other similar response time tasks extant within the PD literature.M = 62.13, SD = 9.59) and 40 healthy participants , and the second consisting of 20 PD patients  and 20 healthy participants . To ensure that no baseline ability differences existed between groups, Wechsler Adult Intelligence Scale (WAIS) full scale IQ estimates were computed for all participants, using the National Adult Reading Test (NART) [M = 24.49, SD = 9.79), and the second sample , with no significant severity differences demonstrated between samples. The spectrum of motor severity within the clinical group is graphically depicted with an area graph in Figures Two independent samples were drawn for this study, the first consisting of 40 PD patients (Age: t (NART) . PatientThe response time tasks used in this study started with an instruction to watch a fixation point (asterisk) in the centre of the computer screen, while depressing the home key (measuring 1.905 cm × 1.905 cm) in the centre of the response console. For the 'uncued' task, participants were not given any advance information concerning the location of the upcoming stimulus. For the 'cued' task, an arrow appeared in place of the fixation point (i.e. in the center of the screen) for a period of 2 seconds, immediately following the disappearance of the fixation point, and correctly cued the location of the upcoming stimulus on all trials. The visual stimulus to which the subject responded was presented on the right or left side of the monitor, at a random interval (between 500 and 1500 ms) following the fixation point ('uncued') or the arrow ('cued'). Participants responded to the stimulus by moving the index finger of their dominant hand from the home key to a response key (measuring 1.905 cm × 1.905 cm) placed 3.175 cm to the left or right of the home key, as directed by the stimulus placement on the screen. The time measured between the onset of the visual stimulus and a participant's movement from the home key was defined as reaction time (RT), and the time measured between a participant's lift from the home key and depression of the response key was defined as movement time (MT). Each task consisted of 10 practice trials, and 40 experimental trials. A participant's RT and MT was computed as the unit-weighted average of scores on the 'cued' and 'uncued' choice response time tasks. This testing apparatus is described in further detail by Johnson et al. .All patients involved in the study were asked to remain drug-free overnight, and to delay taking their morning anti-Parkinsonian medications until after the testing. To avoid any confounding effects resulting from different levels of caffeine intake among participants, all participants were asked to have a normal caffeine-free breakfast prior to testing. None of the participants reported any acute physiological conditions that may have precluded them from putting forth their best effort during the testing session. All procedures and materials were approved by the Health Sciences Research Ethics Board at the University of Western Ontario.In the first sample , separate receiver operator characteristic (ROC) curves were generated for the composite RT and MT scores. The best prediction (i.e. the largest area under the ROC curve) was achieved using the composite MT score. The cutoff score was identified as the point on the curve that maximized sensitivity, with a specificity of at least 70%. This cutoff score was determined to be 230 ms . To control for the possibility that classification success in the first sample was the result of a capitalization on sample-specific variability , this clp < 0.025) were positively correlated with the UPDRS, suggesting that these response time tasks are good predictors of the severity of Parkinsonian symptoms, particularly when considering the MT component of response time.To identify the extent to which response time predicted disease progression, correlations were computed between the UPDRS and the aggregate RT and MT scores. As the samples demonstrated no significant differences on the UPDRS, correlations were computed across all data collected in both samples. Both RT  and MT  differences between PD patients and healthy participants . The resStandardized objective test batteries will be diagnostically useful in two general scenarios: (a) as an adjunct to the physical examination performed by a specialist (to improve diagnostic accuracy), and (b) as a standardized preliminary screening tool, for situations in which a movement disorders expert is unavailable for the physical examination. The latter situation is more important than the former, as primary care physicians are often the first point of contact for these patients. Given the waiting times to see a movement disorders specialist, patients that are considered likely to have PD (based on a screening measure) might be assigned a higher priority in their wait for an initial appointment. This assumes, of course, that primary care physicians will have access to the appropriate motor performance testing devices – and while this is currently prohibitive from a logistical standpoint, it is technologically feasible for the simple tasks described herein to be packaged in smaller (perhaps handheld), less expensive devices.A MT battery may also allow for the communication of results in a "common metric" – without relying on subjective clinical judgments, thereby complementing other clinical tools. Aside from its diagnostic utility, MT batteries may also be useful in tracking a patient's progress as he/she undergoes treatment. At present, motor evaluations conducted during the clinical exam are the only method for tracking change, and this is considerably more qualitative than the MT measures described herein. Along similar lines, MT batteries could make useful adjuncts to clinical drug trial protocols, as they provide good quantitative measures of motor skill that may be used to gauge the effectiveness of the medication under study – in the present study, MT was able to explain 34.81% of the variability in UPDRS motor scores.It should, of course, be noted that the present research was only used to separate PD patients from healthy participants, and so it has not been demonstrated to have any differential diagnostic capabilities . Future research in this area should, therefore. investigate the differential diagnostic power of response time batteries – it may be that the MT battery administered in this study are detecting a general 'impairment' factor, and is not useful as a standalone instrument for the diagnosis of Parkinson's disease. Extending the study base to include patients with disorders such as progressive supranuclear palsy would provide important information concerning appropriate norming that may be done to maximize diagnostic utility.At the very least, however, these results suggest that the use of simple response time batteries may serve as a useful adjunct to other clinical assessment batteries, and may also open interesting avenues of exploration into the consideration of the biological underpinnings of reaction time, and its relationship to movement disorders in general.None declared.AJ conceived and designed the study, collected all data in the norming sample, supervised data collection in the cross-validation sample, supervised data analysis, and contributed to the writing of the paper. PV supervised data collection in the norming sample, assisted in the development of the response time tasks, contributed to the data analysis, and to the writing of the paper. QA assisted with data collection in the cross-validation sample, and contributed to the writing of the paper. LG did all clinical testing on patients in the norming sample. RS assisted with data collection in the cross-validation sample. MJ provided patient diagnoses for participants in the norming sample and the cross-validation sample, and contributed to the writing of the paper.All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Until recently the most accepted technique of doing root canal treatment stresses multiple visit procedure. Most schools also concentrated upon teaching the multi-visit concept. However, it has now been reported that the procedure of single visit treatment is advocated by at least 70% of schools in all geographical areas. It was therefore the aims of the present study to find the incidence of post-obturation flare-ups following single and multiple visit endodontic treatment procedures, and to establish the relationship between pre-operative and post-obturation pain in patients referred for endodontic therapy in a Nigerian teaching Hospital.st, 7th and 30th day. The presence or absence of pain, or the appropriate degree of pain was recorded for each recall visits and the interval between visits. The compiled data were analysed using chi-square where applicable. P level ≤ 0.05 was taken as significant.Data collected included pulp vitality status, the presence or absence of pre-operative, inter-appointment and post-obturation pain. Pain was recorded as none, slight, or moderate/severe. Flare-ups were defined as either patient's report of pain not controlled with over the counter medication or as increasing swelling. The patients were recalled at three specific post-obturation periods, 1Ten endodontic flare-ups (8.1%) were recorded in the multiple visit group compared to 19 (18.3%) flare-ups for the single visit group, P = 0.02. For both single and multiple visit procedures, there were statistically significant correlations between pre-operative and post-obturation pain (P = 0.002 and P = 0.0004 respectively). Teeth with vital pulps reported the lowest frequency of post-obturation pain (48.8%), while those with nonvital pulps were found to have the highest frequency of post-obturation pain (50.3%), P = 0.9.The present study reported higher incidences of post-obturation pain and flare-ups following the single visit procedures. However, single visit endodontic therapy has been shown to be a safe and effective alternative to multiple visit treatment, especially in communities where patients default after the first appointment at which pain is relieved. Until recently the most accepted technique of doing endodontic treatment stresses multiple visit procedures. Most schools also concentrated upon teaching the multi-visit concept. However, it has now been reported that the procedure of single visit treatment is advocated by at least 70% of schools in all geographical areas .Some of the problems of root canal treatment are post-obturation pain, inter-appointment pain and swelling. Although these in most cases do not last long, but could be a source of embarrassment to the dentist and annoying for the patient, more so if the tooth was symptomless before the commencement of treatment. Literature review revealed varied opinions on the incidence and severity of post-obturation pain. Some authors reported slightly more post-obturation pain following single visit than with multiple visit procedures ,3. Otherst, 7th, and 30th post-obturation days, and to compare these results with those reported in previous studies.Endodontic treatment in Nigeria is carried out in the department of restorative dentistry of the four dental schools and in few private dental clinics located in major cities, health centres and general hospitals. Previous report revealed that few cases of root canal treatment were undertaken and that root canal treatment was completed in multiple visits, specifically three visits for about half the teeth treated . ReasonsConsenting patients referred to the department of Restorative Dentistry for root canal therapy within a period of twelve months were randomly assigned for either single visit or multiple visit procedures. For the multiple visit procedures, Patients that defaulted after the first appointment (incomplete treatment) were excluded from the study. For each tooth treated, the clinical factors and conditions existing before, during and after the completion of treatment were recorded. This data included pulp vitality status, the presence or absence of pre-operative pain, post-obturation flare-ups and degree of post-obturation pain. For patients requiring root canal treatment on more than one tooth, the treatment of each tooth was separated by a period of at least four weeks to allow for proper evaluation. The pulp vitality was determined by an electric pulp tester  in combination with the presence of pulpal haemorrhage.st, 7th and 30th day. At each post-obturation recall visit, the patients were interviewed to determine whether or not there were symptoms at the present visit and whether or not there had been symptoms during the interval between the present visit and the previous one. The presence or absence of pain, or the appropriate degree of pain was recorded for each recall visit and the interval between visits. Pain was recorded as none, slight, or moderate/severe. Slight pain was defined as any discomfort no mater how brief in duration that did not require medication and that did not impair masticatory function in any way. Moderate/severe pain was defined as pain requiring medication or other palliative treatment. Impairment of masticatory function (discomfort in chewing) was recorded as moderate/severe pain. Endodontic flare-ups were defined as either patient's report of pain not controlled with over the counter medication and or increasing swelling.The patients were recalled at three specific post-obturation periods, the 1The root canals were obturated with multiple gutta-percha cones and a zinc oxide-eugenol based sealer, using the lateral condensation technique. The compiled data were analysed using chi-square where applicable. P level ≤ 0.05 was taken as significant.st post-obturation day, of these 107 were completed in single visit and 136 were completed in multiple visit. Eighty-six (86) had vital pulps and 157 had nonvital pulp canal contents. Two hundred and twenty seven (227) reported for check-up on the 7th post-obturation day, of these 104 was completed in single visit and 123 completed in multiple visit. Two hundred and twenty two reported for check-up on the 30th post-obturation day, 102 completed in single visit and 120 in multiple visit.Two hundred and eighty three (283) teeth in 255 patients were treated in all, given a ratio of 1.11 teeth per patient. Of these 56 were excluded from the study due to non-availability of patients at post-obturation recall visit. These exclusions were randomly distributed between treatment groups, with no differential loss to follow-up . The treatment groups were fairly comparable, with similar distribution of tooth types between treatment groups, Table 2 = 9.04, p = 0.002). For the multiple visit procedures, 88 teeth presented with pre-operative pain out of which 55 (62.5%) reported post-obturation pain. 48 teeth had no pre-operative pain out of which 6 (12.5%) had post-obturation pain . These show that for both single and multiple visit procedures, there were statistically significant correlations between pre-operative and post-obturation pain , that is patients presenting with pain not controlled by over the counter medication and or increasing swelling, were recorded in the multiple visit group compared to 19 18.3%) flare-ups for the single visit group. This shows a significant difference , Table % flare-uTeeth with vital pulps reported the lowest frequency of pain 48.8%), while those with nonvital pulps were found to have the highest frequency of pain (50.3%), Table 8.8%, whist and 7th days respectively 35.5% and 16.3% were higher than those in the multiple visit group 30.2% and 9.8%. Chi square test indicated no statistically significant differences  whose pulps were non-vital.Many authorities in the field of endodontics advice against the completion of root canal treatment in single visit in order to prevent post-obturation pain, especially in cases presenting with pre-operative pain ,12.In the present study more flare ups occurred in the single visit group (18.3%) than in the multiple visit group (8.1%), showing a disadvantage for single visit treatment at a 95% confidence level, Table . This isPrevious studies have shown that there is a strong positive correlation between pre-operative and post-obturation pain ,14,15. Tth day in both groups, these present a strong indication that practitioners should not overreact to early post-obturation symptoms by immediately initiating endodontic retreatment procedures or extraction of the involved tooth. Apart from the reported higher incidence of flare-ups in the single visit group, the post-obturation pain experienced by the patients in both groups compares favourably well with each other. Therefore the higher incidence should not be taken as condemnation for the single visit endodontic therapy, it should however stress the fact that a thorough understanding of the basic endodontic principles is important in considering each case on an individual basis before making a decision as to whether or not it can be completed in one visit [Despite the high percentages of post-obturation pain reported on the first post-obturation day in both groups Table , seven dne visit . As it iThe present study reported higher incidences of post-obturation pain and flare-ups following the single visit procedures. However, single visit endodontic therapy has been shown to be a safe and effective alternative to multiple visit treatment, especially in communities where patients default after the first appointment at which pain is relieved.The author(s) declare that they have no competing interests.AOO conceived of the study, participated in its design, performed the statistics, and participated in the final write-up of the manuscript. CIU participated in the design, collected the data, drafted the initial manuscript, and participated in the final write-up of the manuscript.The pre-publication history for this paper can be accessed here:


Although percutaneous coronary intervention (PCI) is becoming the standard therapy in ST-segment elevation myocardial infarction (STEMI), to date most patients, even in developed countries, are reperfused with intravenous thrombolysis or do not receive a reperfusion therapy at all. In the post-lysis period these patients are at high risk for recurrent ischemic events. Early identification of these patients is mandatory as this subgroup could possibly benefit from an angioplasty of the infarct-related artery.Since viability seems to be related to ischemic adverse events, we initiated a clinical trial to investigate the benefits of PCI with stenting of the infarct-related artery in patients with viability detected early after acute myocardial infarction.The VIAMI-study is designed as a prospective, multicenter, randomized, controlled clinical trial. Patients who are hospitalized with an acute myocardial infarction and who did not have primary or rescue PCI, undergo viability testing by low-dose dobutamine echocardiography (LDDE) within 3 days of admission. Consequently, patients with demonstrated viability are randomized to an invasive or conservative strategy. In the invasive strategy patients undergo coronary angiography with the intention to perform PCI with stenting of the infarct-related coronary artery and concomitant use of abciximab. In the conservative group an ischemia-guided approach is adopted .The primary end point is the composite of death from any cause, reinfarction and unstable angina during a follow-up period of three years.The primary objective of the VIAMI-trial is to demonstrate that angioplasty of the infarct-related coronary artery with stenting and concomitant use of abciximab results in a clinically important risk reduction of future cardiac events in patients with viability in the infarct-area, detected early after myocardial infarction. Management of acute myocardial infarction (AMI) has underwent considerable changes in the last two decades and the management of patients with AMI has become more established ,2. In paData from large clinical trials indicate that after successful thrombolysis more than 50% of patients have a significant residual stenosis and about 30% of patients have spontaneous or inducible ischemia -9. In thTrials have been performed to study the effect of routine angioplasty on clinical outcome early after AMI treated with thrombolysis -20. MostTo date, non-invasive risk stratification after AMI has mainly focused on exercise stress testing. The inability to exercise, the low diagnostic accuracy and resting ECG abnormalities, however, remain important limitations in the detection of ischemia .Several post-infarction observational studies investigated viability as prognosticator and showed that the presence of viability in the infarct area was highly predictive for future coronary events like recurrent ischemia, recurrent infarction, left ventricular failure, and death -31. ViabIn this context it should be noted that other post-infarction studies have shown viability to be associated with an improved prognosis -36. Two Based on the aforementioned assumptions, we initiated a clinical trial to investigate the benefits of percutaneous coronary intervention (PCI) of the infarct-related artery in patients with viability detected in the early, subacute phase of myocardial infarction. To demonstrate viability, we use low-dose dobutamine echocardiography (LDDE). This test can safely be performed 48 hours after acute MI ,27,30,31We hypothesize that, in order to prevent future cardiac events, PCI is only useful in patients with viability in the infarct zone early after myocardial infarction.Patients admitted to any of the participating centers with an acute or recent myocardial infarction, who are not treated by direct or rescue angioplasty, and who are stable during the first 48 hours after the acute event, are screened for the study. Patients < 80 years of age are considered suitable for the study when they have definite myocardial infarction, as demonstrated by an significant rise in creatine kinase-MB levels , 1 mm ST segment elevation in two or more standard leads or 2 mm ST segment elevation in two contiguous chest leads, and/or the development of Q waves.The criteria for exclusion are: viability testing technically not possible (poor echo-window), contraindications for dobutamine echocardiography (arrhythmia), and coronary angiography , serious life-threatening non-cardiac illness, ECG abnormalities making the evaluation of the ST segment impossible , and an unreliable follow-up.The study complies with the Declaration of Helsinki and all ethics committees of the participating centers have approved the protocol. All eligible patients provide written informed consent.The study is a prospective, multicenter, randomized, controlled clinical trial. In the VIAMI-trial, patients who are admitted to the hospital with an acute myocardial infarction and who did not undergo primary or rescue PCI, are evaluated by LDDE within 3 days of admission. Patients with unequivocal signs of viability in the infarct-area are randomized to an invasive or a conservative treatment strategy. In the invasive strategy patients undergo coronary angiography with the intention to perform PCI with stenting of the infarct-related coronary artery. In the conservative group an ischemia-guided approach is adopted with stress testing before discharge from the hospital. When the test is highly suggestive for ischemia, coronary angiography will be performed. If revascularization is performed, this will be scored as a secondary endpoint. Patients without viability will serve as a registry group with long-term follow-up Fig . These pThe primary endpoint is the composite of death from any cause, recurrent infarction, and unstable angina. The secondary endpoints are need for revascularization, the occurrence of angina pectoris (CCS classification), and the incidence of heart failure (NYHA classification). Left ventricular function is also evaluated as determined by echocardiography at 3 months, 6 months, and 1 year follow-up.A reinfarction is diagnosed if there is an increase in the total creatine kinase and MB isoenzyme activity (2 times ULN) and either a history of ischemic chest discomfort, or electrocardiographic changes indicative for transmural ischemia or necrosis.For the diagnosis of unstable angina, the patient must be hospitalized with ischemic chest pain or discomfort occurring at rest or with minimal exertion. In addition, the need for intravenous medical intervention and/or objective evidence of myocardial ischemia is required. For extensive description of the end points definitions, see Table A clinical endpoints and safety monitoring committee will continuously check and monitor all events in a blinded manner. Reinfarction after revascularization will be scored as described in Table If the inclusion criteria are met and none of the criteria for exclusion, viability testing by LDDE is performed within 3 days of admission.Preferably, beta-blockers are withdrawn 24 hours before the test ,27,30,31Dobutamine is administrated intravenously at doses of 5, 10, and 15 μg/kg/min, for 5 minutes at each dose. When a 10% increase in heart rate is not achieved with 15 μg/kg/min, a 5-minute infusion with 20 μg/kg/min can be used as the final stage of the procedure.Viability is defined as the improvement of wall motion abnormalities in two or more segments of the infarct zone. Changes from hypokinesia to normokinesia and from dyskinesia or akinesia to hypo- or normokinesia are considered an improvement in wall motion abnormality. Dyskinesia changing to akinesia is not considered as an improvement. Patients are continuously monitored by a 12 lead ECG and blood pressure is recorded at the end of each stage. All views are recorded at rest and during dobutamine on VHS videotape. All videotapes are sent to the core-lab  and will be analyzed by 2 experienced observers. A third observer is used in case of disagreement to reach consensus.® by Philips). The images will be displayed side-by-side in a quadscreen format to facilitate the comparison of images.For subsequent off-line analysis, the echocardiographic images will be digitized from VHS videotape and transferred to a working station  will be performed to measure percent diameter stenosis, reference diameter and cross-sectional area stenosis. The degree of stenosis will be determined in the view in which the stenosis is most severe.When feasible, PCI will be performed (with or without etc) when there is a significant (≥ 50%) stenosis or occlusion of the infarct-related coronary artery, with the intention to perform primary stenting of the infarct-related artery, with concomitant use of abciximab, according to the EPILOG protocol  After stPatients will be followed for a period of three years. Follow-up data will be obtained of all patients during visits at the outpatient clinic in the first year, and by telephone interview in the second and third year of follow-up. Left ventricular function (volumes and ejection fraction) is determined by echocardiography at 3 months, 6 months, and 1 year of follow-up.The VIAMI-trial is conducted to investigate the differences in clinical outcome between an invasive and a conservative strategy in patients with demonstrated viability in the infarct-area. The expected event rate in the viability positive group is estimated to be 35 percent. To demonstrate with a power of 80%  that PCI leads to a 50% reduction in event rate in the invasive group compared to the conservative group, 200 patients will be needed in each group. Therefore, we intend to randomize a total of 400 patients in this trial, with interim analysis after 200 included patients.The formal stopping rules for the study are the following: If one of the treatment strategies appears significant superior at interim analysis (P ≤ 0.01), the study will be stopped.t test. Differences between proportions will be assessed by chi-square analysis; a Fisher's exact test will be used when appropriate. Event-free survival curves are computed with the Kaplan-Meier method, and the differences between these curves are tested with a Mantel-Cox log-rank test. A primary endpoint analysis is planned at 30 days, 6 months, and 1 year of follow-up. Subgroup analyses are planned to determine whether the treatment effect is more or less pronounced in certain subgroups. The data will be subgrouped by sex, age, diabetes, anterior infarction, time from onset of symptoms to treatment, and the use of thrombolytics. All analyses will be performed on an intention-to-treat basis. Also, the outcome per-protocol will be evaluated. Such an analysis seems worthwhile, as it will reflect the true influence of PCI on post-thrombolytic ischemic events.Baseline descriptive data will be presented as mean ± standard deviations (SD). Differences in clinical and echocardiographic variables will be assessed by unpaired Student's Enrollment of patients started April 1, 2001. Recently, an interim analysis was performed after the inclusion of 200 patients, having a 30 day follow-up. The Clinical Event Committee recommended continuation of the trial.The VIAMI-trial is the first multicenter, randomized, controlled clinical trial, investigating the clinical benefits of percutaneous catheter intervention of the infarct-related artery in patients with demonstrated viability in the early, subacute phase of myocardial infarction.The authors declare that they have no competing interests.RBvL recruited and analysed all data and drafted the manuscript. GV have been involved in drafting the article and revised it critically. OK and JGFB revised the manuscript critically. CAV and FCV have given final approval of the version to be publiced.


Retroviruses have a diploid genome and recombine at high frequency. Recombinant proviruses can be generated when two genetically different RNA genomes are packaged into the same retroviral particle. It was shown in several studies that recombinant proviruses could be generated in each round of HIV-1 replication, whereas the recombination rates of SNV and Mo-MuLV are 5 to 10-fold lower. The reason for these differences is not clear. One possibility is that these retroviruses may differ in their ability to copackage genomic RNAs produced at different chromosomal loci.To investigate whether there is a difference in the efficiency of heterodimer formation when two proviruses have the same or different chromosomal locations, we introduced two different Mo-MuLV-based retroviral vectors into the packaging cell line using either the cotransfection or sequential transfection procedure. The comparative study has shown that the frequency of recombination increased about four-fold when the cotransfection procedure was used. This difference was not associated with possible recombination of retroviral vectors during or after cotransfection and the ratios of retroviral virion RNAs were the same for two variants of transfection.The results of this study indicate that a mechanism exists to enable the preferential copackaging of Mo-MuLV genomic RNA molecules that are transcribed on the same DNA template. The properties of Mo-MuLV genomic RNAs transport, processing or dimerization might be responsible for this preference. The data presented in this report can be useful when designing methods to study different aspects of replication and recombination of a diploid retroviral genome. Retroviruses are a family of RNA viruses which replicate through a DNA intermediate . The uniReverse transcription entails two DNA strand transfers during minus and plus DNA synthesis. Since the retroviral virion contains two molecules of the viral RNA, the first DNA transfer might be either intramolecular, transferring to the same template, or intermolecular, transferring to the other template. In the model of Spleen necrosis virus (SNV) it was found that the minus-strand DNA transfer is exclusively intermolecular , while aA reason why there are differences in the rates of recombination between HIV-1 and gammaretroviruses (SNV and Mo-MuLV) is not known. It has been suggested that these differences may be associated with the differences in the template switching frequencies of retroviral reverse transcriptases . A recenIn this study, we have investigated whether there is a preference in the formation of homodiploid virions during the mixed retroviral infection. To explore this possibility, we have used the forced recombination system which included two Mo-MuLV-based retroviral vectors containing different selectable markers and one of the vectors having a deletion of the PBS region. These vectors were introduced into the packaging cell line using two different methods, cotransfection, to provide tandem integration, or sequential transfection, and the frequencies of recombination for the vectors have been compared.To study whether there was a preference for the formation of homodiploid virions in the mixed retroviral infection we have used two different methods, cotransfection and sequential transfection, to introduce genetically different retroviral vectors into the host cells. Since plasmid DNA transfected into eucariotic cells is usually tandemly integrated in a chromosome -19, it iIn this study Mo-MuLV-based retroviral vectors were used as partners for recombination. These vectors contained the Mo-MuLV sequences as follows: the 5' and 3' LTRs, ψ region, a part of gag-sequences before XhoI site position 1560 ), and 14560 , andSince pDΔpbsSVpuro RNA is impaired at the initiation of reverse transcription, this function can be restored when the cDNA initiated on the copackaged pDHEneo RNA is transferred to the puro RNA template during the first jump; minus-strand synthesis continues through the puro gene, and the template shift occurs within the leader region. Thus, the restoration of retroviral vector containing the puro gene is possible via homologous recombination with the neo-containing construct at the sequence identity in the leader region of the genome.The experimental scheme employed in this study is outlined in Figure It was expected that plasmid DNA of retroviral vectors pDHEneo and pDΔpbsSVpuro cotransfected into the packaging cell line would be tandemly integrated into the host genome. To study the integration of plasmid DNA, the PCR analysis was performed with the primers hybridizing to the 3' end of neo gene  and to the SV40 early promoter region . Using these primers, the specific PCR products could be obtained if the pDHEneo and pDΔpbsSVpuro are located in the same chromosomal site. On the other hand, PCR products could be generated with only one of the primers when identical molecules of plasmid DNA were integrated in the opposite orientation. However, the efficiency of amplification in this case seems to be very low because such sequences will contain inverted repeats.The PCR analysis was performed using chromosomal DNA prepared from different cell clones generated after cotransfection or sequential transfection of vectors. PCR products were separated by gel electrophoresis, transferred onto nylon membrane and hybridized with 3' neo specific probe. An example is presented in Figure We also used RT-PCR-based assay to examine the ratios of retroviral virion RNA molecules for cell clones generated by different methods of transfection. Since retroviral vectors differed by localization of EcoRI sites in the leader regions, these restriction sites were used as markers to distinguish the two coamplified PCR products obtained with primers specific to this region Figure . EcoRI d3 to 6.3 × 104 CFU/ml and puromycin titers from 5.1 × 101 to 8.0 × 102 CFU/ml. In the cotransfection experiment, the G418 titers varied from 3.1 × 104 to 1.1 × 105 CFU/ml and puromycin titers from 1.4 × 103 to 3.6 × 103 CFU/ml. The frequency of recombination was calculated from the puromycin- and G418-drug-resistant colony titers .The restriction enzyme marker differences in the leader regions of vectors provided a means to analyze the nature of recombinants in NIH 3T3 cells examined by PCR assay. Cellular DNA was analyzed from eight PuroThese experiments demonstrated that the frequency of recombination between vectors localized in the same chromosomal site was about four-fold higher than that of vectors with different chromosomal locations. These data suggest that there might be a preference for the formation of diploid retroviral genome from RNA molecules that are transcribed on the same DNA template. On the other hand, it could not be completely excluded that the high frequency of recombination for retroviral vectors in the cotransfection experiments occurred during or after transfection procedure.To study the possibility of recombination between cotransfected vectors during or after transfection, we used the defective vector in which the 5' LTR promoter was deleted. This vector, pDΔpSVpuro, is almost completely homologous to pDΔpbsSVpuro with the exception of 194 bp in the U3 region . The amplified region of pLTRΔpbs was analyzed by sequencing. The resulting construct, pDΔpbsSVpuro, was generated by exchanging the KpnI-PstI (nucleotide positions 32 to 564) fragment of pDSVpuro with the corresponding fragment of pLTRΔpbs.pMOV9 containing the complete copy of Mo-MuLV provirus and retroviral vectors pDneo and pDSVpuro have been described earlier and were used as the progenitor for all the constructions ,30. pDneequences , neomyciequences . To genepDHEneo is identical to pDneo except the point mutations in the sequences flanking the DLS region. These mutations converted the Mo-MuLV sequences in this region into new restriction sites for HindIII and EcoRI. A description of the cloning steps performed to generate this vector is available upon request.To produce pDΔpSVpuro, the enhancer-promoter sequences of U3 region in pDΔpbsSVpuro were deleted. For this purpose the 3.4 kb SacI-BamHI fragment containing 36 bp of 5' U3 region starting from SacI site and including all other vector sequences of pDΔpbsSVpuro was inserted into the SacI and BamHI sites of pTZ18 plasmid.All DNA manipulations were performed by standard procedures .2, 200 μM each dNTP, 1 % DMSO, 100 nM primers for 20 cycles . The products were separated on 0.8 % agarose gel, transferred onto nylon membrane , and hybridized with neo-specific probe . Probes were generated by the random-primer method with [α32P] dATP [Genomic DNA purification and hybridization were performed by standard molecular techniques . DNA preod with αP dATP [3Virion RNA was purified from filtered culture medium from transfected cells and used in RT-PCR assays . BrieflyNIH3T3 (murine cell line) and GP+envAM12 (amphotropic 3T3-based packaging cell line with MLV Gag + Pol and Env genes)  were groThe cell clones producing transfected vectors were established by transfecting GP+envAM12 cells with vector plasmids using the dimethyl sulfoxide-polybrene method . PuromycViral infection was performed immediately after harvesting the virus. The supernatants were harvested from 90 % confluent stable producer cell clones after 16 hour intervals and filtered through the 0.45 μm filters. Infections were performed in the presence of 8 μg/ml polybrene (Sigma) for two hours at 37°C. Puromycin- and G418-resistant cfu titers were determined using the infection of NIH3T3 cells by end-point dilution.The authors declare that they have no competing interests.SAK carried out most experiments and made substantial contributions to conception and design. AIK and ABO carried out analysis of integrated plasmid DNA by hybridization and participated in the works with cell cultures. IKF conceived of the study, participated in the design and coordination, and drafted the manuscript. All authors read and approved the final manuscript.


Desmoplastic small round cell tumor (DSRCT) is a rare and highly aggressive neoplasm. The cytological diagnosis of these tumors can be difficult because they show morphological features quite similar to other small round blue cells tumors. We described four cases of DSRCT with cytological sampling: one obtained by fine needle aspiration biopsy (FNAB) and three from serous effusions. The corresponding immunocytochemical panel was also reviewed.Papanicolaou stained samples from FNAB and effusions were morphologically described. Immunoreaction with WT1 antibody was performed in all cytological samples. An immunohistochemical panel including the following antibodies was performed in the corresponding biopsies: 34BE12, AE1/AE3, Chromogranin A, CK20, CK7, CK8, Desmin, EMA, NSE, Vimentin and WT1.The smears showed high cellularity with minor size alteration. Nuclei were round to oval, some of them with inconspicuous nucleoli. Tumor cells are clustered, showing rosette-like feature. Tumor cells in effusions and FNA were positive to WT1 in 3 of 4 cytology specimens (2 out 3 effusions and one FNA). Immunohistochemical reactions for vimentin, NSE, AE1/AE3 and WT1 were positive in all cases in tissue sections.The use of an adjunct immunocytochemical panel coupled with the cytomorphological characteristics allows the diagnosis of DSRCT in cytological specimens. Desmoplastic small round cell tumor (DSRCT) is a rare and highly aggressive neoplasm described as a distinct clinicopathologic entity in 1989 by Gerald and Rosai . Other pHistologically, a typical feature of DSRCT is the presence of clusters of tumor cells distributed within a cellular stroma. The shape of clusters varies from round to elongate. Tumor cells are small to medium-sized with round to oval hyperchromatic nuclei, with inconspicuous nucleoli. Necrotic cells and mitosis are common features. Cytoplasm is usually scanty, and cell borders are indistinct. Intracytoplasmic eosinophilic rhabdoid inclusions may be found in larger cells with nuclear pleomorphism . The immThe cytological smears of DSRCT obtained by FNAB are moderately cellular. Tumor cells show round to oval nuclei with fine chromatin and inconspicuous nucleoli. Cytoplasm is scanty to moderate, with variable number of vacuoles. Tumor cells are arranged in loose clusters. Occasionally, spindle fibroblast-like cells are observed. Stromal fragments may be detected . EffusioWe retrieved from the cytological files of Hospital do Cancer – A. C. Camargo four cytological specimens from 3 patients diagnosed with DSRCT, including one fine-needle aspiration sample and 3 fluid samples, during the last five years (2000–2004). FNA was performed on an inguinal mass of one patient. Alcohol-fixed smears were stained with Papanicolaou technique. Serous effusions were prepared with Cytospin . We evaluated two peritoneal fluid samples and one pleural fluid sample. One case  had a cellblock available. All cases were confirmed by histological analysis and immunohistochemical reactions. The histological sections were cut in sections of 4 μm and stained with H&E and immunohistochemistry. Immunocytochemical study was also performed on all cases.Immunohistochemical and immunocytochemical reactions were performed using streptavidin-biotin peroxidase technique with positive and negative controls. Diaminobenzidine was the chromogen. Table 22-year-old white female, with abdominal pain. Video-laparoscopy showed a liver mass and multiple peritoneal implants diagnosed as DSRCT. Six months after the diagnosis, she started chemotherapy for four months, and reduction of tumor mass was observed. One month after the end of chemotherapy, the tumor was removed. Macroscopically, tumor mass measured 5.0 × 4.0 × 3.8 cm and was involving uterus, pericolic tissue, and vagina. Histological analysis shows also involvement of both ovaries and large bowel wall. Ten out of 13 lymph nodes showed metastasis of DSRCT. The peritoneal fluid colleted during surgery was negative for neoplastic cells. Eight months after the first surgery, she presented with a recurrence in the abdominal cavity and a new resection of the tumor mass showed involvement of cecal appendix. Peritoneal fluid sample collected at that time was positive for malignant cells. In the follow up examination, seven months after the second surgery, it was found an inguinal tumor mass of 15 mm. FNA was performed and showed DSRCT metastasis. After the diagnosis, this patient was transferred to another institution.Seven year-old male with back pain and fever. CT scan showed pleural effusion and a mediastinal mass measuring 16.0 × 9.0 cm. Tumour mass showed involvement of soft tissues. Surgical biopsy and pleural drainage were performed. The patient was treated with radiotherapy and chemotherapy, but died 8 months after the diagnosis.Male, 15-year-old had acute abdominal pain and was submitted to an exploratory laparotomy that disclosed a large pelvic mass, involving epiplon and sigmoid, cecum, liver and peri-aortic lymph nodes. This patient had multiple nodules on peritoneal surface. The biopsy of tumor was performed. One month after the diagnosis, chemotherapy was initiated. The patient was submitted to chemotherapy during 8 months, with reduction of more than 50% of tumor mass. A second laparotomy was done to excise retroperitoneal and retrovesical mass. At this time peritoneal fluid sample was collected. After surgery, chemotherapy was continued. The patient is alive, with residual disease.The smears showed high cellularity. The tumor cells exhibited a slight variation in size. Nuclei were round to oval, some of them with small nucleoli. The cytoplasm was scanty. Tumor cells are clustered, with rare clusters showing rosette-like features. The background of the smears showed lymphocytes. Figure .All fluid samples showed similar features. The samples showed high cellularity. Tumor cells were more frequently arranged in tridimentional clusters, but occasionally, isolated cells are also seen. Additionally, clusters showing rosette-like features are rarely observed. Nuclei were round to oval, some of them with small nucleoli. The cytoplasm was scanty Figure .The distribution of immunoreactivity in histological and cytological samples from the patients are summarized in Table the histological sample collected before chemotherapy, exhibited immunohistochemical positivity for vimentin, epithelial membrane antigen (EMA), neuron specific enolase (NSE), chomogranin A, and desmin in dot-like perinuclear pattern. Cytokeratin expression was observed with anti-cytokeratin cocktail (AE1/AE3) and Cytokeratin 8. Tumor cells also expressed WT1 protein.tumor cells exhibited positivity for vimentin, EMA, NSE, chomogranin A, AE1/AE3 and WT1. Desmin and cytokeratins 7, 20 and 34BE12 were negative.tumor cells exhibited positivity for vimentin, NSE, AE1/AE3 and WT1. The immunohistochemical study was performed before chemotherapy.DSRCT is a rare neoplasm that affects young patients. It may present a problem in the differential diagnosis with other small round cell tumors. The diagnosis of DSRCT however can be established with correlation of clinical, cytological and immunocytochemical features. The cytological features that we found in the smears obtained by FNA are similar to other descriptions in the literature. Similar to reports of Zeppa et al , we did One of the characteristics of DSRCTs is its dissemination along serous surfaces. Due to this fact, development of serous effusions is a common clinical finding in DSRCTs patients, with detection of tumor cells in the fluid. In effusions, tumor cells may be present in aggregates but no obviously architectural arrangement is seem.Demonstration of a divergent phenotype and the reciprocal translocation characteristic of DSRCT are critical to the diagnosis.In a reported series of 32 cases of DSRCTs , 88% of The establishment of a specific reciprocal translocation t  as diagnostic in DSRCT was based on the results of Sawyer et al . Shen et22, 23. In addiction to consistent WT1 expression, the typical serosal involvement in DSRCT has raised the possibility that this tumor might be a blastematous tumour derived of primitive mesothelium [EWS gene encodes a protein which the precise function and normal role has not yet been elucidated. Recently, Thomas et al  proposedothelium . Mesotheothelium .We detected WT1 immunoreactivity in all tumors tissues and in 2 out of 3 serous effusions with malignant cells, as well as on FNAB smears. The high frequency of DSRCTs with WT1 protein expression suggests that in consensus with clinical tomographic and cytological findings, this antibody may be used to confirm the diagnosis of DSRCT in cytological samples. We observed a negative WT1 reaction in the cytological sample of patient 3. This sample was collected 10 months after the end of chemotherapy protocol. We can hypothesize if chemotherapy hampered a different antigenic pattern in malignant cells, and influenced this result.Among other small round cell tumors, most of cases of rhabdomyosarcomas and neuroblastomas do not disclose nuclear WT1 staining ,27. CompIn conclusion, cytological and immunophenotypical findings in an appropriate clinical context is sufficient to suggest DRSTC, what sounds highly contributive for us, considering the high aggressiveness of this tumor.