In recent years, outcome prediction models using artificial neural network and multivariable logistic regression analysis have been developed in many areas of health care research. Both these methods have advantages and disadvantages. In this study we have compared the performance of artificial neural network and multivariable logistic regression models, in prediction of outcomes in head trauma and studied the reproducibility of the findings.1000 Logistic regression and ANN models based on initial clinical data related to the GCS, tracheal intubation status, age, systolic blood pressure, respiratory rate, pulse rate, injury severity score and the outcome of 1271 mainly head injured patients were compared in this study. For each of one thousand pairs of ANN and logistic models, the area under the receiver operating characteristic (ROC) curves, Hosmer-Lemeshow (HL) statistics and accuracy rate were calculated and compared using paired T-tests.ANN significantly outperformed logistic models in both fields of discrimination and calibration but under performed in accuracy. In 77.8% of cases the area under the ROC curves and in 56.4% of cases the HL statistics for the neural network model were superior to that for the logistic model. In 68% of cases the accuracy of the logistic model was superior to the neural network model.ANN significantly outperformed the logistic models in both fields of discrimination and calibration but lagged behind in accuracy. This study clearly showed that any single comparison between these two models might not reliably represent the true end results. External validation of the designed models, using larger databases with different rates of outcomes is necessary to get an accurate measure of performance outside the development population. In recent years, outcome prediction studies have become the avante garde in many areas of health care research, especially in critical care and trauma. However acceptable models for outcome prediction have been difficult to develop . AccordiArtificial neural networks (ANNs) are mathematical constructs modeled on interconnection of nodes (neurons) giving a loose association with the animal nervous system. ANNs employ nonlinear mathematical models to mimic the human brain's own problem-solving process. Just as humans apply knowledge gained from past experience to new problems or situations, a neural network takes previously solved examples to build a system of "neurons" that makes new decisions, classifications, and forecasts. ANNs are complex and flexible nonlinear systems with properties not found in other modeling systems. These properties include robust performance in dealing with noisy or incomplete input patterns, high fault tolerance, and the ability to generalize from the input data . Neural Lang EW et al have compared ANN with Logistic Regression in prediction of outcome after severe head injury and concluded that the differences in the results obtained with the two models were negligible .Almost all of the published articles indicate that the performance of ANN models and logistic regression models have been compared only once in a dataset and the essential issue of internal validity (reproducibility) of the models has not been addressed.The objective of this study was to compare the performance of ANN and multivariate logistic regression models for prediction of mortality in head trauma based on initial clinical data and whether these models are reproducible. We used different variables even if they were interdependent.th revision (ICD-10) as well as the outcome of the patients. Data collection was conducted by a group of trained physicians who had completed special training courses to become familiar with the process of extracting Abbreviated Injury Score (AIS-90) codes and filling out the relevant questionnaires. For quality control (QC) purposes each hospital had a physician, who was responsible for overseeing the data gathering process. In the intubated patients GCS were calculated according to the other portions of the scale by this physician. Finally, a medical practitioner examined all the checklists in order to evaluate and amend them if deemed necessary based on pre-arranged and fixed protocols.Among 8452 trauma patients' records admitted to the emergency departments of six major university hospitals in Tehran from 23 August 1999 to 22 September 2000, the records of 1271 patients whose main trauma was head injury, were selected for this study. The selection of head trauma as the main trauma was based on the definition of principal diagnosis in the Uniform Hospital Discharge Data Set (UHDDS). It defines the principal diagnosis as "that condition established after study to be chiefly responsible for occasioning the admission of the patient to the hospital for care". For making determination of the main trauma more practical in the case of ambiguity, hospitalization in the neurosurgical ward was used as an additional guideline. The database was based upon the trauma data registry program began in 1996 in Trauma Research Center, Sina Hospital, a hospital affiliated with the Tehran University of Medical Sciences ), clinicSince we were trying to build and compare models for prediction of outcome mainly based on the initial clinical data, only data related to the GCS, tracheal intubation status, age, systolic blood pressure (SBP), respiratory rate(RR), pulse rate(PR), injury severity score (ISS)(upon admission) and outcome were used in our study. In order to prepare the data for the Neural Network software and to enhance the reliability of the data, three variables of systolic blood pressure, respiratory rate and pulse rate were transformed to dichotomous(1,0) variables. Low systolic blood pressure was defined according to the following cutoff points: up to 5 years of age, less than 80 mmHg; and 5 years of age or older, less than 90 mmHg. Respiratory rate of 35 per min and pulse rate of 90 per min were selected as limits for definition of tachypnea and tachycardia. Other variables including GCS, age, systolic blood pressure (SBP) and injury severity score (ISS) variables were also converted from decimal (Base 10) to binary (Base 2). This conversion was carried out in order to render the input data suitable for processing by our ANN software with its default settings. The data and the data format were similar for both ANN and logistic regression models.The dataset was divided randomly into two sets, one set of 839 cases (66% of the whole dataset) for training and 432 cases for testing the model. A model was built using a training set with logistic regression. GCS, tracheal intubation status(dichotomous), age, SBP(dichotomous), RR(dichotomous), PR(dichotomous) and ISS were the independent variables and the outcome  was the dependent variable. The logistic regression analyses were performed using Intercooled STATA for windows, Version 6 (STATA Corp., College Station, TX) "logistic" default options.The built logistic model was tested using the testing dataset (432 cases). These steps  were repeated 1000 times. This resulted in 1000 pairs of training and testing datasets  which were saved for further processing by the neural networking.The ANN used in this study was a standard feed-forward, back-propagation neural network with three layers: an input layer, a hidden layer and an output layer. The input layer consisted of 23 input neurons, the hidden layer consisted of fifteen hidden neurons, and the output layer consisted of one output neuron Fig. . The leaThe training and testing datasets were the same as those were used with regression models, thus there was an ANN and a logistic model for each training and testing dataset..PDP++ version 3.1 was used for the artificial neural network analysis as it has a powerful built-in scripting language and is freely available. This software can be downloaded from Discrimination and calibration (goodness of fit) were both measured. Discrimination refers to the ability of a model to distinguish those who die from those who survive. A perfectly discriminating model would assign a higher probability of death to all cases that died than to any case that survived. The discriminatory power of the models was analyzed using the area under the receiver operating characteristic curves (ROC-A(z)). ROC curves were constructed by plotting true positives (patients who died and whom the model predicted as dying [i.e. sensitivity]) versus the false positive fraction (fraction of the patients who lived and were incorrectly classified as dying [i.e. (1 – specificity)]).A ROC-A(z) value of one corresponds to a test that perfectly separates two populations, whereas a ROC-A(z) value of 0.5 corresponds to a perfectly useless test that performs no better than chance.The relative calibration of the models, that is, how accurately the models predicted over the entire range of severity, was compared using Hosmer-Lemeshow (HL) statistics. The HL statistics is a single summary measure of the calibration and is based on comparing the observed and estimated mortality for patients grouped by estimated mortality. The resulting statistic follows a chi-squared distribution, with degrees of freedom equal to two less than the number of groups (10 in this study). The smaller the HL statistics, the better the fit, with a perfectly calibrated model having a value of zero.A probability cut point of 0.5 was used to classify observations as events or nonevents. The overall accuracy  of the final model was determined by comparing the predicted values with the actual events.For each of one thousand pairs of ANN and logistic models (trained and tested on the same datasets), these indices (area under the ROC curves, HL statistics and accuracy rate) were calculated and compared using paired T-tests (P < 0.05).All the statistical analyses were performed using Intercooled STATA for windows, version 6 STATA Corp., College Station, TX) and its downloadable add-on ado files.. Further details are available from the corresponding author.Some of the scripts used both for the STATA and PDP++ and the designed neural networks can be downloaded from the site of the Tehran University of Medical Sciences Table As is seen in Table The confidence intervals of the all the measures show higher variation in the ANN models results.Prognostic evaluation of the patients without CT scan findings may have limited applicability considering the availability of CT scans in the majority of the trauma centers. Nevertheless, the idea of prognostic models based on initial clinical data at admission is still worth trying and seems to be of practical value in some situations. Although omission of CT data weakens our armamentarium significantly, patients without paraclinical and imaging data make the real trauma scenes at which medical staff should have an evaluation. Actually as neurosurgeons we have initial clinical judgments based on our growing experiences and in many situations they prove to be true. We can expect computers do similar things and help us. In fact, vague situations like what we see in primary evaluation of head trauma patients are where ANN may prove to be superior to traditional linear modeling. This is one of the reasons that although many paraclinical and imaging factors are known to be of significant predictive value in the outcome of the head trauma patients, this study only used clinical measures which are simply available to a physician in the emergency department.Calculating ISS and AIS are not simple tasks and needs training. This can reduce the practicability of the results. The pupillary size and reactivity are one of the clinical signs with prognostic value that were not considered in the study due to defect of the main database in this regard.As is seen the mortality rate and the percentage of patients with GCS < 8 are the same (7.5%). This is a coincidence, but emphasizes the need for further studies in the populations with different rates of outcome.The mean GCS was 13.5 ± 3. The standard deviation is over the top score of GCS (15) and means that the GCSs were skewed towards higher levels of consciousness.Not considering the exact time interval between head trauma and admission which is simply available for the medical staff is one of the weak points of this study.) somewhat difficult in other situations where pre-hospital care services are more advanced.The lower intubation rate in this population study related to the distribution of the GCS may show the lower quality of the pre-hospital care prevailing in the hospitals selected for this study. This may render the reproduction of the results using the same network , and seem to promise "prediction" without the difficulties associated with use of mathematics.ANNs are rich and flexible nonlinear systems that show robust performance in dealing with noisy or incomplete data and have the ability to generalize from the input data. They may be better suited than other modeling systems to predict outcomes when the relationships between the variables are complex, multidimensional, and nonlinear as found in complex biological systems.The difficulty in developing models using artificial neural networks is that there are no set methods for constructing the architecture of the network. The most common type of artificial neural networks is the feed-forward back propagation multiperceptron (used in this study).Another limitation of neural network models is that standardized coefficients and odds ratios corresponding to each variable cannot be easily calculated and presented as they are in regression models. Neural network analysis generates weights, which are difficult to interpret as they are affected by the program used to generate them . This laSeveral early applications of neural networks in medicine reported an excellent fit of the ANN model to a given set of data. The impressive results usually were derived from over fitted models, where too many free parameters were allowed. Linear and logistic regression models have less potential for overfitting primarily because the range of functions they can model is limited.Neural network models require sophisticated software. The complexity and unfamiliarity of ANN has been a major drawback of this technique so far. However, as palmtop computing becomes increasingly powerful and popular, the complexity of ANNs may become less onerous in real-time clinical settings. Furthermore, there are some theoretical advantages comparing a predictive ANN model over conventional models such as logistic regression.One such advantage is that ANN model allows the inclusion of a large number of variables . AnotherAlthough, one of the strengths of ANNs is their ability to still find patterns despite missing data, in this study a dataset with no related missing values was used.Recently the task of comparison between these two models has been addressed from different points of view. Considering the publication bias, several published works in the medical literature have demonstrated the success of the ANN approaches. In a review carried out by Sargent on 28 major studies, ANN outperformed regression in 10 cases (36%), was outperformed by regression in 4 cases (14%) and the 2 methods had similar performance in the remaining cases. Sargent concluded that both methods should continue to be used and explored in a complementary manner. Gaudart et al. using simulated data, have compared the performance of ANN and linear regression models for epidemiological data and concluded that both had comparable performance and robustness and despite the flexibility of connectionist models (like ANN), their predictions were stable. This study was primarily designed to compare the performance of an ANN and a multivariable logistic regression analysis with the goal of developing a model for predicting the outcome in head injury and for studying their internal validity (reproducibility). Also setting up a standalone practical model for prediction of mortality in the head trauma patients was a secondary goal of this study.Using freely downloadable software (PDP++) and making the networks and scripts accessible by the researchers can be perceived as an advantage of this study.This study showed that ANN models significantly outperformed logistic models in both senses of discrimination and calibration, although lagged behind in accuracy. It is pointed out that the calibration values should be treated with some caution in this study, since according to Hosmer and Lemeshow,  in descrThis study clearly shows that in a single comparison of these models based on the same data there is 22.2% chance of getting discrimination results contrary to our findings in majority of comparisons. This ratio is 43.6% for calibration and 32% for the accuracy results. These figures are practically important and imply that any single comparison between these two models cannot reliably represent their final performance.Although considerable efforts, through many trial-and-errors, were made to optimize the design of the network, the designed ANN models could, and should, be further improved. In line with any other predictive models, likewise the findings of this study need to be externally validated. The networks are downloadable and the results can be studied in other study populations with divergent data and different survival ratios.So far, there is no single algorithm that performs better than all other algorithms for any set of given head injury data. To this end, there is room for much more work to be done before a definite conclusion can be reached.Should the results be reproducible in other populations, using a simple preprogrammed calculator (or other programmable computing devices) and minimal training of the personnel, this model and similar ones may emerge to be of considerable practical value in triage of the patients. At that time a dedicated instead of general purpose ANN software should be designed for this purpose.The authors concur with the conclusion arrived by Tu  that logIn conclusion, this study compared models for the prediction of outcome in head injury using trauma data from hospital registries in Tehran, the data was applied to artificial neural network and multivariable logistic regression analysis. The predictive ability of the artificial neural network model was found to be comparable to that of the logistic regression model. Specifically, the ANN models significantly outperformed logistic models in both senses of discrimination and calibration but lagged behind in accuracy. Although the performance of the models were studied when the models were applied to the different samples of the original population study, external validation is necessary to get an accurate measure of performance outside the development population. Studies using larger databases with different rates of outcomes may further clarify the differences between artificial neural network and logistic regression models in head injury outcome prediction and their clinical implications.The author(s) declare that they have no competing interests.BE carried out the data extraction, performed the analysis and drafted the manuscript. KM supervised the analysis and critically reviewed the statistical viewpoints. HEA, MG and EK supervised the study and participated in its coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


There is limited population-based data on diet and physical activity behaviors and weight loss among users of prescription weight loss medications. Most findings are from clinical settings or from research that includes organized behavioral programs.We analyzed data from the 1998 Behavioral Risk Factor Surveillance System, an annual telephone survey conducted in all fifty states, the District of Columbia and Puerto Rico. The sample consisted of 135,435 noninstitutionalized adults aged 18 years old and older. We determined the prevalence and odds of prescription weight loss medication use, odds of 10% weight loss, and among current weight loss medication users, the prevalence and odds for diet and physical activity behaviors.10.2% of obese women and 3.1% of obese men reported using prescription weight loss medications in the past 2 years. Of users, 28.2% had lost at least 10% of their pretreatment body weight. The odds of losing at least this much weight were higher among women, those who usually consumed ≥ 5 fruits and vegetables daily and those who met physical activity recommendations. Among current prescription weight loss medication users, 26.7% reported both eating fewer calories and meeting recommended leisure-time physical activity levels (<40% of any group met both). Of those meeting both recommendations, almost half (47.2%) had lost 10% of their pretreatment body weight. Of current users, 9% reported using the medications for weight maintenance.Only 26.7% of prescription weight loss medication users reported following recommended diet and physical activity behaviors. Further research is needed to assess whether behavioral changes are associated with greater weight loss and maintenance among prescription weight loss medication users. Lifestyle modifications, including behavior therapy, diet, and physical activity, are the cornerstone of weight management . CurrentThe initial goal of weight loss therapy is to reduce body weight by approximately 10% over a 6-month period ,4. WeighUsing population-based data we estimated that 4.6 million American adults used prescription weight loss medications between 1996 and 1998 . During We examined data from the 1998 Behavioral Risk Factor Surveillance System (BRFSS). The BRFSS is a telephone survey of health practices that is conducted each year by all state health departments. Each state, the District of Columbia, and Puerto Rico select an independent probability sample of noninstitutionalized residents aged 18 years and older. In 1998, 149,806 persons responded to the BRFSS; several investigators have published detailed descriptions of survey methods and quality control indices . The aveTo determine prescription weight loss medication use, respondents were asked, "In the past two years, have you taken any weight loss pills prescribed by a doctor? Do not include water pills or thyroid medications." Responses were coded as: 1) "Yes, I am currently taking them", 2) "Yes, I have taken them but I am not currently taking them", or 3) "No, I have not taken them".Respondents with a positive response were asked about their prepill weight, "How much did you weigh just before you started taking prescription weight loss pills for the first time?"At the end of the interview, respondents were asked to report their current height and weight without shoes. We used current weight and pretreatment weight to calculate current and pretreatment body mass index (BMI), respectively. BMI was calculated as weight (either current or pretreatment) in kilograms divided by current height in meters squared. BMI was categorized into 3 groups: normal weight (<25.0), overweight (25.0–29.9), and obese (≥ 30.0) . PercentWe used the following questions to determine weight control strategy, "Are you now trying to lose weight?" Those who responded "no" were then asked, "Are you now trying to maintain your current weight, that is to keep from gaining weight?" Additional questions on dietary practices and physical activity were asked of the subset of respondents (95,100) who responded "yes" to trying to lose or maintain their body weight. These respondents were asked "Are you eating either fewer calories or less fat to lose/maintain weight?" Their responses were either: 1) "Yes, fewer calories", 2) "Yes, less fat", 3) "Yes, fewer calories and less fat", or 4) "No". They were also asked "Are you using physical activity or exercise to lose/maintain weight? Their responses were either "yes" or "no".All individuals were also asked "In the past 12 months, has a doctor, nurse, or other health professional given you advice about your weight?" Responses were "Yes, lose weight", "Yes, gain weight", "Yes, maintain current weight", "No". In an earlier section individuals were asked, "About how long has it been since you last visited a doctor for a routine checkup? The latter question was used to distinguish those who had not received advice due to not seeing a doctor for a routine checkup in the past 12 months.For respondents who were currently using prescription weight loss medication and were trying to lose weight or maintain their current weight, we attempted to determine if they were meeting the minimal lifestyle recommendations. Respondents who stated they were eating fewer calories – either as fewer calories or as both fewer calories and less fat – were classified as meeting minimal dietary recommendations. Respondents were asked about the two physical activities or exercises they engage in most often and about the frequency, duration, and distance (as appropriate) of each activity . ResponsWe also requested demographic and socioeconomic information such as age in years, sex, race/ethnicity, marital status, educational level, and household income. Of the 149,806 respondents, we excluded those who did not report information on prescription weight loss medication use n = 1561), had a missing current weight (n = 5229) or height (n = 1369), were pregnant (n = 1780), were missing current weight goal (trying to lose weight or trying to maintain weight) (n = 749), or were missing information on sociodemographic variables, fruit and vegetable intake, or physical activity (n = 3595). We excluded an additional 88 respondents that had a questionable reported weight, height or BMI that was outside the sex-specific reference values from the Third National Health and Nutrition Examination Survey, 1989–1994 [61, had aWe used SAS and SUDAAN for the statistical analysis to account for the complex sampling design ,17. StudThe results of our multivariate analyses show that the reported use of prescription weight loss medications in the past 2 years was higher among women (4.0%) than men (0.9%). For women, the odds of use was 19% lower among those aged 35–54 years and 75% lower among those 55 years old than among those aged <35 years. The odds of prescription weight loss medication use was 34% lower among non-Hispanic black women than among non-Hispanic white women; and 53% and 39% lower among those women residing in the Northeast and Midwest regions of the United States, respectively, than among women residing in the West region , one tenth lost no weight (women: 12.9%, men: 13.4%), one-tenth lost between 1% and 5% (women: 12.7%, men 21.0%), and one fifth lost between 5% and 9% of body weight (women: 19.6%, men: 24.1%), data not shown.Additionally among users, one-third of women (35.0%) and one quarter of men 25.0%) reported a 10% weight loss, Table 5.0% repoWe also assessed professional advice, diet, and physical activity among current prescription weight loss medication users by current weight intention Table . AlthougOnly one-quarter (26.7%) of current prescription weight loss medication users met both lifestyle recommendations – eating fewer calories and attaining recommended physical activity levels. Our results indicate that among current users, the odds of meeting both recommendations was 62% lower among men than among women and 56% lower among other race/ethnic groups than among non-Hispanic whites Table . We alsoIn a secondary analyses we assessed prevalence of a 10% weight loss among current prescription weight loss medication users who were trying to lose weight (not maintain) by whether or not recommendations were met for eating fewer calories and physical activity (n = 237). 39.6% (SE 5.3) of individuals who reported both eating fewer calories and who met physical activity recommendations had lost at least 10% of their pretreatment weight; 36.5% (SE 6.4) who reported eating fewer calories but who did not meet the physical activity recommendations had lost at least 10% of their pretreatment weight; 54.3% (SE 9.8) who reported not eating fewer calories but met physical activity recommendations had lost at least 10% of their pretreatment weight; and 25.5% (SE 5.9) who did not meet either lifestyle recommendation had lost at least 10% of their pretreatment weight, (chi-square p = 0.03).Although in 1998 clinical guidelines recommended that prescription medication for obesity be used in conjunction with lifestyle modifications [Although patterns of prescription medication use differed by sex, for both men and women the odds of use was higher among those who were obese compared to those who were overweight although at different magnitudes. Our data show than more overweight women used prescription weight loss medications (4.88%) than did obese men (3.11%). It is possible that pharmacotherapy prescribing patterns differ by sex since more women than men attempt weight loss  or that Eating fruits and vegetables and taking part in recommended levels of physical activity through leisure-time activities were more frequent behaviors among prescription weight loss medications users who had lost at least 10% of their body weight. Although we had no direct measurements of caloric intake, fruits and vegetables in their natural state are low in energy density. Thus, adding or substituting fruits and vegetables for energy-dense snacks and sweets can impact satiety and weight gain ,18,19. OAbout 40% of current prescription weight loss medications users who both reduced calories and met leisure-time physical activity recommendations had lost 10% of their body weight. Our data also show that respondents who consumed usually less than 5 fruits and vegetables per day were less likely to meet both diet and physical activity recommendations than did those who consumed 5 or more fruits and vegetables a day. Because fewer than half of physicians counsel obese patients about weight control ,22 we beThe current study had several limitations. Although the participants self-reported pre-medication body weight and current weight, we did not ask for the date prescription weight loss medication use began and it could not be established whether weight loss was solely attributable to pharmacotherapy usage. We were also unable to determine if respondents had lost a substantial amount of weight but then regained the weight (weight history). Self-reported weights and weight history may also be a concern because individuals, especially those who are overweight, may underreport their weight . We lackAlthough we found that about one fifth of participants had lost between 5% and 9% of their pretreatment body weight and one third of participants had lost at least 10% of their pretreatment body weight, the pill dosage and length of medication use were not collected. Thus, some users may have used pills for a short time, such as less than 6 months, and would not be expected to have lost 5% to 10% of their body weight.In current practice, physicians treating obesity can prescribe phentermine, sibutramine, and orlistat (available April 1999). Analysis of national data on patient visits found that phentermine was the most common antiobesity medication in 2001 and in early 2002 (based on January through March figures) , suggestMost nonsurgical obesity treatments lead to weight loss for the first 6 months followed by regain . Thus, sWe found that one in ten obese women and one in 33 obese men reported using a prescription weight loss medication in the past two years. Among medication users, one-third of women and one quarter of men reported a 10% weight loss. Only one quarter of current medication users reported the minimal diet and physical activity behaviors recommended for weight loss. Ongoing population-based surveillance is needed to assess the prevalence of prescription weight loss medication use and whether pharmacotherapy users are making appropriate lifestyle changes in order to lose or maintain weight.BRFSS: Behavioral Risk Factor Surveillance SystemBMI: body mass indexOR: odds ratio95% C.I.: 95% confidence intervalThe author(s) declare that they have no competing interests.The questions that were asked of all authors:Have you received reimbursements, fees, funding, or salary from an organization that may in any way gain or lose financially from the publication of this paper in the past five years? No.Have you held any stocks or shares in an organization that may in any way gain or lose financially from the publication of this paper? No.Do you have any other financial competing interests? No.Are there any non-financial competing interests you would like to declare in relation to this paper? No.HB performed the statistical analysis and drafted the manuscript.LKK conceived the study questions, provided statistical input, and edited the manuscript.MKS conceived the study questions, provided statistical input, and edited the manuscript.All authors read and approved the final manuscript.


Peritoneal dialysis (PD) is the preferred dialysis modality in children. Its major drawback is the limited technique survival due to infections and progressive ultrafiltration failure. Conventional PD solutions exert marked acute and chronic toxicity to local tissues. Prolonged exposure is associated with severe histopathological alterations including vasculopathy, neoangiogenesis, submesothelial fibrosis and a gradual loss of the mesothelial cell layer. Recently, more biocompatible PD solutions containing reduced amounts of toxic glucose degradation products (GDPs) and buffered at neutral pH have been introduced into clinical practice. These solutions contain lactate, bicarbonate or a combination of both as buffer substance. Increasing evidence from clinical trials in adults and children suggests that the new PD fluids may allow for better long-term preservation of peritoneal morphology and function. However, the relative importance of the buffer in neutral-pH, low-GDP fluids is still unclear. In vitro, lactate is cytotoxic and vasoactive at the concentrations used in PD fluids. The BIOKID trial is designed to clarify the clinical significance of the buffer choice in biocompatible PD fluids.The objective of the study is to test the hypothesis that bicarbonate based PD solutions may allow for a better preservation of peritoneal transport characteristics in children than solutions containing lactate buffer. Secondary objectives are to assess any impact of the buffer system on acid-base status, peritoneal tissue integrity and the incidence and severity of peritonitis.® solution for a period of 10 months. Patients will be monitored by monthly physical and laboratory examinations. Peritoneal equilibration tests, 24-h dialysate and urine collections will be performed 4 times. Peritoneal biopsies will be obtained on occasion of intraabdominal surgery. Changes in small solute transport rates, markers of peritoneal tissue turnover in the effluent, acid-base status and peritonitis rates and severity will be analyzed.After a run-in period of 2 months during which a targeted cohort of 60 patients is treated with a conventional, lactate buffered, acidic, GDP containing PD fluid, patients will be stratified according to residual renal function and type of phosphate binding medication and randomized to receive either the lactate-containing Balance solution or the bicarbonate-buffered Bicavera Peritoneal dialysis (PD) is the preferred dialysis modality in children. Advantages of PD over hemodialysis relevant to pediatric patients include its compatibility with a normal lifestyle and full psychosocial integration, the continuous mode of blood purification without dysequilibrium conditions, the absence of vascular access issues and the avoidance of puncture pain. However, the major drawback of PD is its limited technique survival. Almost fifty percent of adult as well as pediatric PD patients must switch to hemodialysis within 4 to 5 years of treatment ,2. While® (Fresenius), a purely bicarbonate-buffered biocompatible PD solution, with that of a conventional acidic, lactate buffered solution by a three-month crossover trial in children on automated PD [® treatment, which was readily reversible when patients returned to conventional solution. This effect was also observed with lactate- or lactate/bicarbonate-buffered biocompatible PD solutions [® was used. Two studies observed a slightly lower initial increase of the functional peritoneal surface area during a single PD dwell with pH-neutral compared to acidic solutions compatible with reduced peritoneal capillary recruitment; this trend was significant in one study [® than with lactate buffered conventional fluid despite identical content of base equivalents.In recent years, a new generation of more biocompatible PD fluids has been introduced into clinical practice. The separation of alkaline and acidic fluid compartments in pluri-chamber bags permits to sterilize glucose at very low pH with greatly reduced GDP formation and yet produce pH-neutral final dialysis solutions, using lactate and/or bicarbonate as a buffer. We recently compared the safety and efficacy of Bicaveramated PD . We obseolutions ,15, and olutions ,16 we obne study -19. FiscWhile these results are encouraging with respect to the long term preservation of the peritoneal membrane and strongly favour the primary use of low-GDP, neutral-pH biocompatible PD fluids, the relative importance of the buffer system is still unclear. In vitro data suggest that lactate per se may compromise local cell functions independently of pH by affecting the cellular redox state and reducing cellular energy sources ,20-22. BThe European Pediatric Peritoneal Dialysis Study Group (EPPS) plans a prospective, randomized study with administration of pH neutral, low-GDP PD solutions containing either lactate or bicarbonate buffer over a period of 10 months.primary objective is to evaluate the effect of lactate vs. bicarbonate buffer on peritoneal transport capacity in children. The hypothesis to be tested is that bicarbonate based PD solutions may allow for a significantly better preservation of peritoneal transport characteristics (D/PCrea) in children compared to a solution containing lactate buffer. Secondary objectives will be to assess differential effects of lactate and bicarbonate buffered PD fluids on acid-base status, surrogate parameters of peritoneal biocompatibility and local and systemic carbonyl stress, peritoneal morphology, the incidence and severity of peritonitis, statural growth and nutritional status. Moreover, this study will be used to assess genetic determinants of the peritoneal transporter status and the evolution of peritoneal morphology over time.The 2) and the type of phosphate binder therapy , since these variables may affect the overall efficacy of metabolic acidosis control. Following stratification, subjects will be randomized centrally to receive either the lactate-containing Balance solution or the bicarbonate buffered Bicavera® solution for a period of 10 months. Both during the run-in period and during the intervention phase, patients will be monitored by monthly clinical and laboratory examinations, including capillary blood gas analyses. In addition, peritoneal equilibration tests (PETs), 24-hour dialysate and urine collections and intraabdominal pressure assessments will be performed at time of randomization  and after 3, 6 and 10 months of treatment (with the study solutions). Also, peritoneal biopsies will be performed on occasion of intraabdominal sugery or laparoscopy prior to start and after termination of the study .This is a multicenter open-labelled, controlled, randomized clinical trial, designed to test the effects of the buffer substance in biocompatible PD fluids on peritoneal small solute transport capacity. All subjects will undergo a 2-month run-in period, in which they receive conventional lactate buffered, acidic, GDP containing PD fluid. During this period, patient eligibility for the trial will be verified and the dialysis dose will be optimized if necessary to ensure appropriate PD adequacy. At the end of this period, the patients will be stratified according to residual renal function , peritoneal neoangiogenesis (VEGF), fibrotic activity (TGF-β) and local inflammation (IL-6). With the same intention, the evolution of peritoneal histomorphology will be assessed in all patients available for sequential biopsies. Moreover, possible differential effects of lactate and bicarbonate buffer on the control of metabolic acidosis will be assessed by monthly blood gas analyses. Finally, the incidence and clinical course of peritonitis will be recorded as a possible indirect marker of local peritoneal macrophage function.2 body surface area, 4) the most recent episode of PD-associated peritonitis, if any, occurred more than 3 weeks ago, 5) signed informed consent by parent/guardian, with a subject aged > 7 years also signing an age-appropriate assent form.Criteria for inclusion in the study are 1) patients above 1 month and less than 19 years of age, 2) end-stage renal disease with manual or automated continuous peritoneal dialysis as maintenance treatment modality, 3) a fill volume approximately of 1100 ml/1.73 mCriteria for exclusion from the study are 1) reduced efficiency of peritoneal dialysis due to anatomic anomalies or intraperitoneal adhesions, 2) uncontrolled hyperphosphatemia, 3) severe pulmonary, cardiac, hepatic or systemic disease including any kind of malignancy, and 4) current or recent (within 30 days) exposure to any investigational drug.Reasons for permanently discontinuing the study medication are 1) renal transplantation, 2) switch to hemodialysis due to PD technique failure, 3) patient/parent withdrawal of consent of participate, 4) patient moving out of the area to a location with no participating center within reasonable distance, and 5) a severe adverse event.study fluids is given in Table ® and Balance will be available in three different glucose concentrations to meet individual ultrafiltration requirements. The fluids will be administered at a dose of approximately 1,100 ml/m2 body surface area per dwell. Both in patients on CAPD and CCPD, the number of cycles and dwell times can be varied according to clinical needs. The dose of dialysis will be tailored individually in order to ascertain a minimum total weekly Kt/VUrea of ≥ 2.0.The composition of the concomitant medication during the run-in period and the study period will be documented in the case report form with respect to type, dosage and mode of delivery. In case of peritonitis (defined by cloudy effluent, white blood cell count greater than 100/mm3 with more than 50% polymorphonuclear leukocytes), treatment will be given intraperitoneally according to international pediatric guidelines [Any kind of idelines  using ceBicarbonate supplementation will be discontinued at start of the run-in period and only re-instituted if blood bicarbonate levels drop below 17 mmol/l despite sufficient dialysis efficacy. The recommended dosage is 0.5 mmol/kg/day divided into 3 doses.adverse event is defined as any untoward medical occurence in a patient who takes the study medication. It does not necessarily have a causal relationship with this treatment. This may be an unfavourable and unintended sign, symptom or disease, which is observed after exposure to the study medication, whether or not considered related to the treatment. Moreover, the participating investigators will report all treatment-emergent adverse events that are observed on the online adverse event form. This applies regardless of the clinical significance or the assessment of study drug causality. In this trial, such adverse events may include inflow pain, severe changes of the state of hydration, abnormal electrolyte and glucose blood levels, peritonitis, abdominal hernia, and allergic reactions.An severe adverse event or reaction is any untoward medical occurrence that a) results in death, b) is life-threatening, c) requires inpatient hospitalization or prolonges an existing hospitalization, or d) results in persistent or significant disability/incapacity.A Any serious adverse event, whether or not considered related to the study medication, and any unexpected drug reactions with significant hazard to the patient population will be reported to the responsible safety assessor at Fresenius Medical Care by phone or by fax within 24 hours following first knowledge of the event. Alternatively the clinical monitor may be informed. This information will be forwarded to and evaluated by the safety monitoring committee, and reports of serious adverse reactions will be disseminated to all participating centers for submission to their respective institutional review boards. Fresenius Medical Care is responsible for passing on the information to relevant supervisory authorities.Data acquisition will be entirely through the internet. The case report form menus will be used to record the following: 1) baseline clinical patient information, 2) physical and biochemical examination variables, 3) study and concurrent medications, and 4) adverse events.Periodic computerized audit reports will be run to monitor data quality and completeness. The data base is stored on a server drive that is backed up to tape daily by Tel-A-Vision, Media Networking GmbH.In order to insure confidentiality, data from each patient will be recorded in the computer data base with a unique contributing center code, study code, sequence number and patient initials. Patient names are never entered online or forwarded in any other form to the coordinating office.Cr from the time of randomization to the conclusion of the 10-month study period. In a previous trial we demonstrated a 6% increase of D/PCr in children on lactate-buffered PD fluid in contrast to a 4% decrease in D/PCr using bicarbonate buffered fluid within 3 months of exposure, resulting in a statistically significant 10% difference in the evolution of peritoneal creatinine transport rate [Cr at least 7 ± 10% can be expected when Balance and Bicavera® are applied for 10 months. To detect this difference with a sensitivity of 80% and an error probability of 5%, at least 15 patients per randomization group will be required. Assuming a 50% drop-out rate due to renal transplantation and other reasons in the course of the study, 60 patients will have to be enrolled.The primary study outcome is the change in 4h-D/Port rate . AssuminA computer based randomization protocol will be generated and applied centrally at the end of the run-in period. Baseline comparability between the two treatment groups will be evaluated with respect to entry criteria. Chi-square and t-tests will be used to assess differences between the two groups on the baseline variables. Any variables that are found to be discrepant between the two groups and that are related to the outcome variables will be treated as co-variates in later analyses.Cr ratios, two strategies will be employed:In order to evaluate the patients' change in D/P1. Repeated measure ANOVA will be performed on those patients who complete the 10-month observation period on the study medications.Crea ratio during the 10-month period. All patients started on study medication will be available for this analytical approach.2. The Kaplan-Meier method will be used to estimate the time in which patients are likely to display a ≥ 7.5 % increase in the D/PThe same strategies will be used to analyze secondary outcome variables.All publications will be authored by members of the scientific advisory committee. Co-authors will have contributed to the design, analysis, execution and actual reporting of the study.Steering committee: C.P. Schmitt, F. Schaefer, K.E. Bonzel, K. RönnholmScientific advisory committee: M. Almeida, K. Arbeiter, G. Ardissino, K.E. Bonzel, A. Edefonti, M. Fischbach, K. Haluany, J. Misselwitz, Markus J. Kemper, K. Rönnholm, F. Schaefer, C.P. Schmitt, S. WygodaSafety monitoring committee: V. Schwenger, U. Querfeld, G. OffnerWe report the protocol of a randomized clinical trial designed to test the effect of the buffer type on the evolution of peritoneal tissue integrity and transport function in children treated with 'biocompatible', i.e. neutral-pH, low-GDP PD solutions. The study utilizes the availability of two novel biocompatible PD solutions manufactured by the same company which differ selectively in the buffer employed, namely either pure lactate or pure bicarbonate. The comparative administration of these solutions will provide information on differences in peritoneal cell viability, tissue morphology, local host defense and solute transfer capacity potentially inferred by cytotoxic and/or vasocative effects of lactate administered to the abdominal cavity in unphysiological concentrations.This investigator-initiated trial was designed exclusively by the members of the EPPS scientific advisory committee. None of the investigators have any financial relationship with the manufacturer of the study medication. Financial support is received from Fresenius Medical Care to cover coordination costs and investigator meetings.The pre-publication history for this paper can be accessed here:


This report describes a large international chocolate-associated Salmonella outbreak originating from Germany.Salmonella (S.) Oranienburg isolates were subtyped by the use of pulsed-field gel electrophoresis (PFGE).We conducted epidemiologic investigations including a case-control study, and food safety investigations. S. Oranienburg notifications was registered in Germany. Simultaneously, an increase in S. Oranienburg infections was noted in other European countries in the Enter-net surveillance network. In a multistate matched case-control study in Germany, daily consumption of chocolate , having shopped at a large chain of discount grocery stores , and consumption of chocolate purchased there  were associated with illness. Subsequently, two brands from the same company, one exclusively produced for that chain, tested positive for S. Oranienburg. In two other European countries and in Canada chocolate from company A was ascertained that also contained S. Oranienburg. Isolates from humans and from chocolates had indistinguishable PFGE profiles. No source or point of contamination was identified. Epidemiological identification of chocolate as a vehicle of infections required two months, and was facilitated by proxy measures.From 1 October 2001 through 24 March 2002, an estimated excess of 439 Salmonella-containing products. Particularly in diffuse outbreak-settings, clear associations with surrogates of exposure should suffice to trigger public health action. Networks such as Enter-net have become invaluable for facilitating rapid and appropriate management of international outbreaks.Despite the use of improved production technologies, the chocolate industry continues to carry a small risk of manufacturing  Salmonella spp. is a substantive cause of human gastroenteritis in many parts of the world [Salmonella reports (incidence:94/100,000) received at the federal level by the Robert Koch-Institut (RKI) accounted for 31% of all notifications for the 54 notifiable conditions [Salmonella enterica subspecies enterica serotype Enteritidis (S. Enteritidis) is the predominating serotype followed by S. Typhimurium. They represented 65% and 23% of the reported cases of non-typhoidal salmonelloses with known serotype in 2001. Thus, the remaining ~250 serotypes reported to the RKI in that year, including S. Oranienburg, accounted for only 12%.Non-typhoidal he world . In Germnditions . SalmoneSalmonella and Other Enteric Pathogens (NRC) in Hamburg noted an unusual increase in the number of S. Oranienburg isolates received in October. At that time, no increase was noticeable in the national database for statutorily reportable infectious diseases; 50 S. Oranienburg notifications (median: 1 per week) had been registered for 2001. On November 19, the NRC in Wernigerode informed the RKI that it had received a S. Oranienburg isolate in September. The isolate was submitted by a private laboratory for serotyping and had come with the additional source information "confectionery sample". Upon inquiry, a large German chocolate manufacturer (company A), which produced a broad variety of chocolates and products made thereof, called the RKI on November 27, and confirmed that it had sent in the confectionery sample. According to company A, the positive sample originated from an in-house control of a chocolate product and the pertaining batch, due to be exported to the United States, was completely destroyed and not distributed. Notwithstanding, the number of statutory S. Oranienburg notifications had sharply increased and continued to rise. This report describes the epidemiologic, food safety, and microbiological investigations of this outbreak.In mid-October 2001, the National Reference Center for S. Oranienburg infections about chocolate consumption in the seven days before disease onset, and also to send any remaining chocolate to a food safety laboratory.A standard exploratory questionnaire was distributed on 20 November 2001 via state health departments to all local health departments to aid the collection of data on food and environmental exposure from cases. In addition, local health departments were asked to immediately interview patients with newly reported A request was distributed to participants of the Enter-net surveillance network  on DecemS. Oranienburg isolates from patients and from the in-house chocolate control were found to be indistinguishable by pulsed-field gel electrophoresis (PFGE). On the same day, a multistate case-control study was initiated and coordinated by RKI to test the hypothesis that at least one product from company A was associated with S. Oranienburg-infections. As we were denied a product list from company A, we resorted to the company's web-site and included in our food history evaluation all the products listed there. Some products from company A, e.g., bars of chocolate (brand A), were exclusively sold at a large chain of discount grocery stores (chain X). We found that the majority of chocolates sold at chain X were produced by company A. Therefore, for the analysis we constructed a variable for chocolate(s) purchased at chain X ("chain-X-chocolate") as a proxy for chocolate-products from company A because most patients could remember the flavor of the purchased chocolate, but seldom the brand name.On December 3, while exploration of patients were ongoing and results inconclusive, S. Oranienburg in other countries [S. Oranienburg infection to a public health department before December 6. Case-patients were excluded from the analysis, if they could have been secondary, i.e., if they reported to having had contact with a person with diarrhea in the seven days prior to symptom onset. Cases were selected from the national reportable database by simple random sampling; in Lower Saxony an attempt to interview all case-patients was launched. Case selection was done irrespective of whether patients also had been interviewed with an exploratory questionnaire. At least one age and telephone exchange-matched control subject was selected for each case-patient by sequentially adding 2 to each case-patients' telephone number. Control subjects were eligible if they were in the same age-group as their matched case-patient (0–5 years, 6–17 years, 18–59 years, 60 years or older), had no gastrointestinal symptoms after 1 October 2001, and had not traveled abroad in the seven days prior to the onset of symptoms of the matched case. Telephone interviews were conducted by state health departments, local health departments, and the RKI. Data were analyzed with Epi Info V6.04c (Centers for Disease Control and Prevention, Atlanta, GA).The hypothesis-testing questionnaire collected data on the consumption of chocolates, and some other foods, particularly those previously associated with outbreaks of ountries -5. Food-Salmonella positive chocolate leftovers and five chocolates from grocery stores using the most probable number technique [The local food safety authority inspected company A's production facility and took samples from already packaged ("in-house") chocolates, and from ingredients from its suppliers. Beginning December 11, a nationwide chocolate sampling of German chocolates in grocery stores was initiated by the Federal Ministry of Consumer Protection, Food and Agriculture, and was assisted by the Federal Institute for Risk Assessment (Bundesinstitut für Risikobewertung, "BfR"). On December 18, when a chocolate leftover from brand A tested positive, the investigations were tailored to German chocolates from company A. The BfR examined quantitatively four echnique .S. Oranienburg isolated from stool specimens were sent to the NRC from laboratories in Germany and, on Enter-net request, from other countries. Furthermore, isolates from chocolates were submitted from state or private food laboratories in Germany as well as from Canada and the Czech Republic. PFGE-analysis was carried out according to Prager et al [For comparison by the use of PFGE, er et al .S. Oranienburg up to reporting week 42, but 462 reports in the following 23 weeks (15 October 2001–24 March 2002, "outbreak period", figure S. Oranienburg reports were registered assuming a background rate of one report per week. The median age was 15 years (range: 0–92 yrs), 240 (52%) patients were female. There was no difference in the gender distribution within the single 10-year age-bands (P = .51). All 16 states of Germany reported S. Oranienburg cases during the outbreak period, with the highest incidence in the state of Schleswig-Holstein (1.78/100,000) bordering on Denmark. In total, 206 of the 440 German counties were affected with a median of one report and a maximum of 16 from the city of Hamburg during the outbreak period.In 2001, the RKI received 50 reports of S. Oranienburg.Sixty exploratory questionnaires were received from eight states by the end of 2001. Forty-three (88%) of 49 patients with information on chocolate consumption had a symptom onset after 1 October 2001. Of the 34 who gave information as to where they bought the chocolate, 21 (62%) explicitly reported chain X. Some reported exclusively having eaten chocolate bars from brand A, among them a two-year-old child. On 18 December 2001, two months after the initial outbreak alert, a leftover consumed reportedly by this child in the seven days before symptom onset tested positive for S. Oranienburg had been reported in Denmark from October 18 through December 10, compared with only two cases in 2001 before October 18. None of the clustered cases were travel-related [S. Oranienburg problem, independently suspected German chocolate bought in chain X as the source of the Danish outbreak. Chocolate was the only food item that all patients reported eating. The majority stated purchasing chocolate in chain X, which, although German, operates internationally [S. Oranienburg infections was reported from other countries such as Austria, Belgium, Finland, Sweden, The Netherlands  after disease onset in case-patients. Twelve case-control pairs were excluded from chocolate-specific analysis, nine because the case-patients could have been secondary, one due to illness in September, and two where the control subjects could not remember whether they had eaten chocolate. Of the 48 cases and 50 controls that were analyzed, 24 (50%) case-patients and 32 (67%) control subjects were female, 22 (46%) case-patients were younger than 10 years. Ten (21%) of the case-patients reported to have suffered from bloody diarrhea and 14 (29%) were hospitalized  of 381 chocolates that were tested and reported to BfR during the outbreak period. S. Oranienburg was isolated from two different brands of company A; all positive chocolates were produced during the same week in August 2001. Estimates of the number of Salmonella in the tested chocolates ranged between 1.1 and 2.8 per gram.The local food safety authority in Germany did not identify hygienic deficiencies at the production facility. Samples obtained in the beginning of December 2001 from in-house chocolates (n = 12), as well as from cocoa (n = 3) and cocoa powder (n = 7) from a supplier of company A tested negative. This applied also to German chocolates sampled in grocery stores until 18 December (on that day a leftover tested positive). Overall, S. Oranienburg isolates from human cases of gastroenteritis originating in Germany (n = 52), Austria (n = 19), Belgium (n = 8), Canada (n = 6), Denmark (n = 4), The Netherlands (n = 4), Sweden (n = 4), and the Czech Republic (n = 1). Furthermore, 15 chocolate isolates were sent to the NRC for PFGE-analysis from Germany (n = 12), Canada (n = 2), and the Czech Republic (n = 1). They came from an in-house sample, from leftovers of chocolates consumed by patients in their incubation period, and from chocolates sampled in grocery stores in Germany. The PFGE profiles of S. Oranienburg isolates from patients with symptom onset after 1 October 2001 (outbreak period) in Germany and in the other countries mentioned above, except Canada, were indistinguishable  in the week prior to symptom onset, but not in the seven days before the interview. Case-patients were more likely than control subjects to report eating chocolate daily, likely indicating an increased probability of having been exposed to contaminated chocolate. Furthermore, patient isolates from the outbreak period shared a PFGE profile with isolates from chocolates but differed from isolates of patients who became sporadically diseased with S. Oranienburg before the outbreak. In addition, the food histories and microbiological results from S. Oranienburg patients in several other countries pointed to the same source [We describe an international outbreak of e source ,10.Salmonella infections after consumption of contaminated chocolate, although rare, have been known since the 1960's [Salmonella have been recovered from chocolates in these outbreaks, suggesting a very low infectious dose. Estimates of the number of S. Oranienburg cells per gram in this outbreak ranged from 1.1–2.8. However, we cannot exclude that the bacteria were unevenly distributed in the chocolate(products) and that those parts carrying many viable cells were not tested quantitatively. In chocolate, the low moisture (water activity aw: 0.4–0.5) and high sugar content does not favor bacterial growth, but significantly increases thermal resistance [Salmonella against the acidic conditions of the stomach [e 1960's . Common e 1960's -17  can carry Salmonella spp., the low water activity and high fat content in chocolate increases thermal resistance so that temperatures reached during chocolate production  do not  a small number of Salmonella may be sufficient to cause disease, even with low-level contamination, chocolate can affect large number of persons (often children) scattered over a wide area, and thus, has the potential to cause serious public health consequences.It is noteworthy that the case-control study did not identify further products as risk factors. This applied also to the second contaminated brand of company A, which was included in the recall. The small proportion of study cases (25%) mentioning having eaten chain-X-chocolate lends support to the hypothesis of more contaminated brands (even from other manufacturers), which could be one explanation for the continuing case-occurrence. However, inaccuracies due to lack of brand awareness may have played a particular role in this outbreak, and the time-delay between disease onset and interview (median: 37 days) may have contributed to an inaccurate recall of cases and their guardians. Furthermore, cases with a disease onset in 2002 may have occurred as a result of a diminished impact of the public warning due to the Christmas season. For example, chocolate gifts received or given for Christmas may not have been thoroughly enough checked for best-before dates stated in the public warning.Identification of vehicles in foodborne outbreaks can become difficult if the exposure is common. Consumption of a wide variety of German chocolates was reported by all case-patients (and 88% of explored patients), but also from 86% of the control subjects in the week prior to onset of illness. When groups are (nearly) universally exposed or a more specific hypothesis cannot be tested, often the best one can do is to establish a "dose-response-relationship" , i.e., uS. Eastbourne outbreak in Canada/USA in 1973/74, 286 environmental samples and 98 chocolate samples from the production-line were examined. No in-line chocolate sample tested positive and overall only 6 (1.6%) samples were positive  were obtained. In a An Enter-net urgent inquiry was sent after the first results of molecular subtyping suggested a link between human cases and chocolate from company A. Until then, investigators in Germany and Denmark had worked independently unaware that the outbreak extended outside of their respective countries. An earlier inquiry, ideally as early as an outbreak was suspected by the investigating countries (or as an increase was noted in the Enter-net database), may have speeded up hypothesis generating, and thus, may have helped in earlier identification of the vehicle, thereby preventing illnesses.Finally, a public warning or recall of company A products did not occur before a brand A leftover tested positive although the confluence of information – the results of the case-control study, the Danish investigations, and the subtyping comparison between human isolates and the in-house sample – had already pointed to company A products as the source of the outbreak. Yet, no specific product or lot had been identified at the time. For this reason, a recall or a public warning were considered excessive responses by the German food safety authority. However, relying on microbiological confirmation in leftovers, if available for testing, is disputable  and is dangerous in unopened food packages because critical time can elapse before a positive culture in food is obtained . TherefoSalmonella-containing products. For the future, awareness among German food safety authorities must be heightened for the need to base public health action not exclusively on laboratory confirmation in food, and to conduct timely and comprehensive source investigations to enhance food safety in the long-run. The international scale of this outbreak shows how easy it is to distribute a contaminated product across many countries. This underlines the necessity of mechanisms for international surveillance and information dissemination such as Enter-net to ensure that international outbreaks can be dealt with rapidly and in an appropriate manner. Similar networks should be set up or, if existing, should be connected (possibly overseen by WHO), to allow rapid communications to other parts of the world when it is clear that a contaminated product is distributed internationally.To our knowledge, this is the largest reported chocolate-associated outbreak, the seriousness being emphasized by the hospitalizations (29%) and self-reported bloody diarrhea (21%) of the study cases. Despite the use of improved production technologies, the chocolate industry continues to carry a small risk of manufacturing The author(s) declare that they have no competing interests.Salmonella in chocolate. EW coordinated food safety investigations in this outbreak. AE conducted the Canadian part of this outbreak investigation. AS conducted the Finnish part of this outbreak investigation. YA conducted the Swedish part of this outbreak investigation. MHK was instrumental in the design of the case-control study. AA coordinated the German part of this outbreak investigation, broadened the scope of this outbreak by prompting an urgent Enter-net inquiry, and helped in designing the case-control study and drafting the manuscript.DW was the principal investigator of the German part of this outbreak; he carried out the statistical analysis of the case-control study, and drafted the manuscript. JD and FF were responsible for the outbreak investigation including the case-control study for the state ("Bundesland") of Lower Saxony. UvT was responsible for the outbreak investigation including the case-control study for the state ("Bundesland") of Northrhine-Westfalia. GF was responsible for the outbreak investigation including the case-control study for the state ("Bundesland") of Hamburg. SE conducted the Danish part of this outbreak investigation. AMH was responsible for the outbreak investigation including the case-control study for the state ("Bundesland") of Hesse. PR detected the outbreak and conducted microbiological investigations. RP and HT conducted the PFGE-analysis. ISTF coordinated the Enter-net inquiries and investigations. SB conducted interviews in the case-control study, designed the database and helped in the analysis. EB conducted quantitative analysis of All authors participated in revising the manuscript.The pre-publication history for this paper can be accessed here:


Epidemiological studies on the potential role of surgery in Creutzfeldt-Jakob Disease transmission have disclosed associations with history of specific surgical interventions or reported negative results.Within the context of a case-control study designed to address surgical risk of sporadic Creutzfeldt-Jakob Disease in Nordic European countries (EUROSURGYCJD Project), a strategy was adopted to categorise reported surgical procedures in terms of potential risk of Creutzfeldt-Jakob Disease acquisition. We took into account elements of biological plausibility, either clinically or experimentally demonstrated, such as tissue infectivity, PrP expression content or successful route of infection.We propose a classification of exposed tissues and anatomic structures, drawn up on the basis of their specific putative role as entry site for prion transmission through contact with surgical instruments that are not fully decontaminated.This classification can serve as a reference, both in our study and in further epidemiological research, for categorisation of surgical procedures in terms of risk level of Creutzfeldt-Jakob Disease acquisition. Case-control research on the association between surgery and sporadic Creutzfeldt-Jakob Disease (CJD) encompasses six studies and one meta-analysis -8, whichIn this section, we summarily describe the on-going study, review reports that possibly shed light on the biological plausibility of surgery-related CJD transmission, and propose principles for re-classification of surgical procedures.The on-going case-control study, entitled "Surgery and risk of Creutzfeldt-Jakob Disease" (EUROSURGYCJD), constitutes a Concerted Action funded by the EU Research Commission, contract QLG3-CT-2002-81223. The main objective of this study is to quantify a putative excess risk of CJD associated with surgery. A secondary objective is to establish a basis for the design of preventive strategies. The most relevant methodological characteristics are: case-control design; exposure measurement prior to disease onset, registered as codes for surgical procedures; matched 5:1, randomly chosen population controls and random sample of population controls. The population base is the resident population in Denmark, Finland and Sweden, covered by the respective hospital in-patient registers. Cases are individuals with diagnoses corresponding to ICD-9 codes 046.1 and 331.5 and ICD-10 code A81.0 at death or at hospital discharge for the period 1987–2002, identified from the respective national hospital in-patient registers and corresponding national surveillance units. A questionnaire will be mailed to the heads of the registered hospital department or surveillance unit, and a copy of the medical record will be obtained for diagnosis validation. Approximately 300 patients will fulfil criteria for definite or probable sporadic CJD, and constitute the study cases. Population controls are: 1) 5 × 1 controls (approximately 1500), matched to the corresponding case by age, sex, county of residence of the case ; and 2) a 20/million sample of the 1987–2002 resident population aged >40 years, randomly selected from the corresponding national population registers.Individual person-numbers will be used for each resident case or control. Diagnoses and surgical procedures at hospital discharge of the corresponding CJD case at any registered time before date of death will be obtained from the three national hospital discharge registers in Sweden, Denmark and Finland. Perusal of surgical records might be undertaken for selected associations in order to understand transmission mechanisms and interpret results. Open-care surgery or dentistry will not be studied. Surgical procedures coded in registers as per national or NOMESCO classifications will be re-classified in accordance with putative levels of transmission risk based on scientific evidence/plausibility.The quality of CJD diagnoses will be assessed by a review of medical records. The accuracy of surgical history given by the registers will be assessed by comparison with that of a sample of controls and surrogate respondents obtained by interview.Centralised data analyses will be conducted by the Spanish team. In specific instances, risk due to blood transfusion, whether or not performed during surgical procedures, might also be studied.Surgery may be a pathway for patient-to-patient transmission of sporadic Creutzfeldt-Jakob Disease (CJD). In many invasive surgical procedures, non-disposable surgical instruments come in contact with tissues that are known to be infective in CJD patients. These same instruments may retain a considerable level of infectivity after routine sterilisation, and in successive patients can come into contact with tissues that may act as entry sites for CJD transmission. Among the almost 300 recorded cases of iatrogenic transmission of CJD, 5 cases have been attributed to surgical instruments employed in neurosurgical procedures, whilst 2 additional cases were caused by the use of a contaminated intracerebral EEG electrode . To dateIn the above-mentioned studies -7, surgiSc) is a conformational isoform of PrPC, a normal host protein present in neurons and other cell types. In sporadic and familial transmissible spongiform encephalopathies (TSEs), "spontaneous" conversion of PrPC into PrPSc is the key pathogenic event, followed by the accumulation, deposition and further conversion of PrPSc in tissues, together with its propagation along specific neural pathways. In the case of transmitted TSEs -when these are not due to direct inoculation into the CNS- a peripheral phase of neuroinvasion by PrPSc is followed by a subsequent phase of prion replication and propagation along the peripheral nervous system, with final access to the central nervous system [Sc in mucosae-associated lymphoid tissue. For the purpose of classifying tissues susceptible to prion inoculation, the following considerations can be derived from this pathogenic model: i) a tissue can act as entry site for prion transmission if it normally expresses PrPC; ii) the level of PrPSc expression of an infected tissue correlates positively with the risk of prion acquisition by that tissue; and, iii) all tissues involved in the propagation chain of infection from peripheral tissues to the central nervous system can act as entry sites for prion transmission.According to the "protein only" hypothesis , pathoges system . In scraSc by Western blot. This three-level classification correlates quite closely with the distribution and levels of PrPC expression in normal nervous and non-nervous tissues in mammals.[Sc detection have been obtained exclusively in animal TSEs and/or vCJD. This is the case of the small bowel, large bowel (including enteric nerve plexuses), adrenal tissue, pancreas and bone marrow.The recently published WHO classification of tissue infectivity in TSEs , though  mammals. The WHO C under normal conditions. This is the case of anterior ophthalmic tissues, skeletal muscle, peritoneum, and subcutaneous tissue rich in sensitive nerve fibres. These anatomical structures have therefore been independently added to our classification as putative routes of entry, with a lower level of risk compared to the high level represented by the central nervous system, sensitive ganglia and posterior eye tissues. The fact that PrPSc has been recently found in 1/4 skeletal muscle samples of sCJD cases[Further relevant data for tissue classification derive from iatrogenic CJD cases and from experimental transmission of prion diseases to animals. In roughly half of iCJD cases the entry site has been the CNS or the eye , whilst in the other half, injection of pituitary hormones means that a peripheral route of entry has to be assumed . ExperimCJD cases promptedA final classification of entry sites for putative surgical transmission of CJD contains tissues, including all those showing positive results for sporadic and familial CJD in the WHO classification , with miWe propose a list intended to be used to generate attributes of single, well-defined surgical procedures and criteria for their classification in terms of putative risk level of transmission. The two main attributes will be: 1) use of non-disposable surgical instruments; and, 2) exposure during surgery of tissues included in the preceding list. High- and lower-risk surgical procedures will be defined by exposure of tissue corresponding to the respective risk level during surgery. In addition, the lowest risk level is represented by those surgical procedures where disposable surgical instruments are not employed or where no listed tissue or anatomical structure is exposed to surgical instruments.A categorisation of surgical procedures based on the above attributes is inevitably prone to some degree of subjectivity, something that should be minimised by adequate methodological assessment. However, this drawback is more than offset by the possible benefits of identifying specific surgical procedures that pose a significant risk of CJD transmission, in terms of increased study power and control of misclassification of exposure by the removal of surgical procedures, which are probably irrelevant for CJD transmission, from gross anatomical classifications of surgery. We are also aware of the fact that whereas the same surgical instruments are commonly employed in an homogeneous group of procedures in some countries, as is the case of Nordic countries, the same instruments may circulate through a much wider range of procedures and putative risk levels in other countries. Final risk for disease transmission in each surgical procedure combines postulated risk related to tissues exposed in that procedure with the highest risk level of tissues to which instruments have been previously exposed. Accordingly, results should always be interpreted in the light of a known or assumed pattern of instrument circulation between surgical procedure groups. Finally, it is worth stressing that the aim of the approach presented here is exclusively to produce a useful tool for epidemiological research in CJD transmission. Under the present state of knowledge, no consequences for the possible adoption of any practical recommendation concerning surgery or further preventive measures are to be drawn from this approach.The author(s) declare that they have no competing interests.AR assumed the basic task of reviewing literature, proposing tissues and structures, and drafting the first manuscript version. JPC indicated the subject domain, suggested differences between disease transmission and disease acquisition as seen from experimental and observational epidemiological research, generated first paragraphs relating to epidemiological aspects. KM suggested changes in epidemiological aspects. ÅS provided some criticisms. MC gave diverse comments, particularly on biological plausibility. HL clarified the need for methodological refinement in future work. All authors read and accepted the final version.The pre-publication history for this paper can be accessed here: