Gcm2−/− mutants. However, the developmental ontogeny and cellular identity of these “thymic” PTH–expressing cells is unknown. We found that the lethality of aparathyroid Gcm2−/− mutants was affected by genetic background without relation to serum PTH levels, suggesting a need to reconsider the physiological function of thymic PTH. We identified two sources of extra-parathyroid PTH in wild-type mice. Incomplete separation of the parathyroid and thymus organs during organogenesis resulted in misplaced, isolated parathyroid cells that were often attached to the thymus; this was the major source of thymic PTH in normal mice. Analysis of thymus and parathyroid organogenesis in human embryos showed a broadly similar result, indicating that these results may provide insight into human parathyroid development. In addition, medullary thymic epithelial cells (mTECs) express PTH in a Gcm2-independent manner that requires TEC differentiation and is consistent with expression as a self-antigen for negative selection. Genetic or surgical removal of the thymus indicated that thymus-derived PTH in Gcm2−/− mutants did not provide auxiliary endocrine function. Our data show conclusively that the thymus does not serve as an auxiliary source of either serum PTH or parathyroid function. We further show that the normal process of parathyroid organogenesis in both mice and humans leads to the generation of multiple small parathyroid clusters in addition to the main parathyroid glands, that are the likely source of physiologically relevant “thymic PTH.”In mammals, parathyroid hormone (PTH) is a key regulator of extracellular calcium and inorganic phosphorus homeostasis. Although the parathyroid glands were thought to be the only source of PTH, extra-parathyroid PTH production in the thymus, which shares a common origin with parathyroids during organogenesis, has been proposed to provide an auxiliary source of PTH, resulting in a higher than expected survival rate for aparathyroid  Due to the important role of PTH in the regulation of physiological activities, disorders in PTH production can cause many diseases in humans. Thus it is very important to understand where PTH is produced and how it is regulated. Many people have been found to have ectopic and supernumerary parathyroid glands without clear ontogenesis. In addition, the thymus, which develops together with the parathyroid during embryogenesis, has been proposed to be an auxiliary source of PTH with endocrine function; however, PTH is also a tissue-restricted self-antigen expressed by the thymus. In this paper, we provide insights into the ontogeny and function of thymus-associated PTH. We found that ectopic and supernumerary parathyroid glands originate from the normal developmental process underlying the separation of parathyroid and thymus, resulting in misplaced parathyroids close or attached to thymus. In the thymus, thymic epithelial cells can produce a low level of PTH via a different mechanism than the parathyroid and provide functional data that TEC-derived PTH does not have endocrine function. In summary, our data show that the thymic source of PTH has no endocrine function and, instead, has an expression pattern in the thymus consistent with that of a self-antigen for negative selection. Parathyroid hormone (PTH) produced by the parathyroids acts as the key endocrine regulator to modulate the physiological actions in the bone, kidney and the intestine to maintain the homeostasis of ionized calcium and inorganic phosphorus concentrations in the extracellular environment. Failure of calcium and phosphorus homeostasis, which can result from PTH production disorders, causes serious physiological consequences in human Mammals have evolved an integrated system consisting of the parathyroid glands, bone, kidney and the intestine, to regulate ionized calcium and inorganic phosphorus homeostasis in the extracellular environment Gcm2 null mouse mutant phenotype identified the thymus, a primary lymphoid organ, as an auxiliary source of circulating PTH in addition to the parathyroids in mice Gcm2 mouse mutants was called into question by the phenotype of Pth null mutants, which can survive in the complete absence of PTH The parathyroid glands were long thought to be the sole source of PTH production and secretion. However, analysis of the aparathyroid rd pharyngeal pouches rd and 4th pharyngeal pouches are thought to give rise to four parathyroids rd pharyngeal pouches, while the pair of superior parathyroid glands (not present in mice) develop with the ultimobranchial bodies from the 4th pharyngeal pouches Despite their distinct primary functions, the parathyroid and thymus organs have a close relationship during organogenesis, initially developing from two shared parathyroid/thymus primordia originating from the bilateral 3Gcm2 mutant mouse reported that these mice were aparathyroid from embryonic stages The original analysis of the Gcm2−/− mice caused a significant decrease of thymic PTH expression but still left a low level of thymic PTH expression, which we identified as originating from mTECs expressing PTH in a Gcm2-independent but Foxn1-dependent manner. Our results indicate that mTEC-derived PTH is not secreted into the general circulation and does not function as a backup mechanism of parathyroid glands, but may function as a self-antigen for negative selection. We further show that the lethality associated with Gcm2 mutation is not related to the presence of thymic PTH or serum PTH levels. Our results also have implications for the molecular mechanism of promiscuous gene expression of tissue-restricted self-antigens in mTECs. Our data also provide an explanation for the origin of ectopic parathyroid adenomas that are often associated with human hyperparathyroidism.To clarify the ontogenesis, regulation of PTH expression, and physiological role of extra-parathyroid PTH-expressing cells, we studied parathyroid and thymus organogenesis in the mouse. We showed that clusters of ectopic parathyroid cells between the parathyroid and thymus or attached to the thymus resulting from incomplete separation of these two organs during normal organogenesis. Analysis of parathyroid organogenesis in human embryos showed a similar phenomenon. Absence of these misplaced parathyroid cells in the thymus in Gcm2−/− mutants on the C57BL/6J and 129/C57BL/6J F1genetic backgrounds for survival and parathyroid function. We found that Gcm2−/− mutants on a C57BL/6J genetic background had a nearly 100% lethality rate . Variations in PTH levels have been reported between C3H/HeJ and C57BL/6 mice, including change in PTH levels in response to altering the calcium content of the diet, as well as differences between strains in BMD, calcium absorption, serum calcium, and calcitriol levels Gcm2 mutants, we designed experiments to determine the ontogenesis of extra-parathyroid PTH-expressing cells. Since the parathyroids and thymus arise from the same embryonic structure, we tracked the process by which the parathyroid and thymus domains resolve into separate primordia in mice using in situ hybridization for Pth and Gcm2. At E12, Gcm2/Pth expression in the parathyroid/thymus common primordia specifically marked the anterior/dorsal Gcm2-positive parathyroid domain with a clear interface at the posterior/ventral Foxn1-positive thymus domain Gcm2/Pth-positive parathyroid domain had started to separate from the thymus domain, and some parathyroid cells were located outside the major parathyroid domain  have detached from the pharynx. Throughout week 7, clusters of Gcm2 expressing cells were located in the anterior portion of the common primordium (4/4 embryos) and at the posterior tip (1/4 embryos) of the migrating elongated thymic structure  and a smaller Gcm2 expressing accessory parathyroid (red arrowhead) associated with a single thymic lobe  (Gcm2 expressing cells are left attached to the upper cordlike thymic structure (Dissected parathyroids and thymic lobes from one side of early and mid week 8 embryos showed three major  week 6) . In addi week 6) , althoug week 6) . FurtherGcm2−/− mice could still amplify Pth at high cycle numbers , while those at the posterior tip of the thymus primordium correspond to the Gcm2-positive clusters at the posterior in week 7. However, it is also possible that these both anterior parathyroids arise from the 3rd pharyngeal pouch and that the 4th pouch-derived parathyroid is no longer associated with the thymus primordium and therefore is not dissected out along with the thymus. In our view, the question of which of the parathyroid rudiments present in human fetal development give rise to the inferior and superior parathyroids in the adult also remains open, and will require further detailed study for its resolution.In our view, it is not yet possible to definitively determine the relationship of the four GCM2-positive structures at week 8 to those present at late week 7, since the thymus and parathyroid primordia are actively migrating at this stage in development and lineage tracing studies are not possible in human embryos. The two GCM2-positive structures associated with the anterior end of the thymus primordium at week 8 may correspond to the major parathyroid foci present at late week 7 .Our original interest in this project was piqued by the differences in lethality between the Pth expression level in mTECs and a Foxn1-dependent and Gcm2-independent pathway for PTH expression in mTECs, more consistent with the terminal differentiation model; Gcm2-dependent PTH in the thymus came exclusively from misplaced parathyroid cells. Microarray analysis indicates that PTH expression in mTECs is Aire-independent Two models have been proposed for the mechanism that regulates the promiscuous expression of TRAs in mTECs. The progressive restriction model proposes a mosaic of TRA expression in immature mTECs characteristic of multi-lineage differentiated cells of endoderm-derived organs, and expressed by the same tissue-specific regulators as in their ‘normal’ tissues All experiments using mice were carried out at UGA with the approval of the UGA Institutional Animal Care and Use Committee. First and second trimester human fetuses were obtained in collaboration with the Reproductive Biology Unit, Little France, Edinburgh. Ethical approval for use of human fetal tissue was granted by the Lothian University Hospitals NHS Trust and the Lothian Research Ethics Committee: Smith et. al. ‘Isolation and propagation of fetal stem cells’ LREC/2002/6/15. Consent was obtained from all donors, and the tissue was anonymized before being made available for research. Use and disposal of tissues are strictly regulated in accordance with conditions stipulated in the Ethics approval and in the University of Edinburgh Health and Safety regulations regarding use of human tissue. All experiments using human tissue were performed at the University of Edinburgh.Gcm2 null mutant has been described Gcm2 mutant mice on a 129/SvEv-C57BL/6J genetic background were backcrossed to C57BL/6J mice for more than 5 generations. These majority C57BL/6J Gcm2 mutant mice were then backcrossed to 129S6 mice (Taconic) to obtain 129S6/C57BL/6 F1 hybrids.The generation and genotyping of Foxn1-nude mice (Jackson Labs) and R26YFP reporter mice Foxn1 was previously described Gcm2;Foxn1 double mutants was done by mating Gcm2+/−; Foxn1nu+/ males with Gcm2+/−; Foxn1nu+/ females. A total of 182 one month old mice from Gcm2+/−; Foxn1nu+/ mating were genotyped at weaning. Reduced survival of genotypes homozygous for the Gcm2 mutation was significant using the chi-square test. Since there were no survival defects detected in Gcm2+/− or Foxn1nu+/ heterozygous mice, we combined heterozygous mice with wild-type mice as a control group.Analysis of In all crosses, for calculating the % survival, the survival of wild-type mice was set at 100%. For staging of embryos, noon on the day of the vaginal plug was designated as E0.5.Embryos were staged according to the standard head/rump measurement and classified according to Carnegie stages. Embryos used in this study were from week 6 (Carnegie stage 16–17), week 7 (Carnegie stage 18–19) and early to mid-week 8 (Carnegie stage 20–21). Embryos were fixed in 4% PFA for 24 hours and stored at −20°C in 100% methanol until used for analysis.5′-TGGAATCCTGTGGCATCCATGAAAC-3′, β-actin reverse 5′-TAAAACGCAGCTCAGTAACAGTCCG-3′, Pth forward 5′-CTGCAGTCCAGTTCATCAGC-3′, Pth reverse 5′-AAGCTTGAAAAGGTAGCAGCA-3′, Gcm2 forward 5′-CATCAATGACCCACAGATGC-3′, Gcm2 reverse 5′-GGCACTTCTTCTGCCTTCTG-3′, Foxn1 forward 5′-TGACGGAGCACTTCCCTTAC-3′, Foxn1 reverse 5′-GGGAAAGGTGTGGGTAGGTC-3′, Gcm1 forward 5′-TGAAAAACAAGCCCTTCAGC-3′ and Gcm1 reverse 5′-TCTGGCTTTGTCACAGATGG-3′. Both Gcm2 and Pth RT-PCR products were confirmed by sequencing.Isolation of RNA and RT-PCR were performed as described in situ hybridization for Gcm2 and Pth was performed as described µm sections were hybridized with digoxigenin-labeled RNA probes at 0.5 µg/ml. Alkaline phosphatase-conjugated antidigoxigenin Fab fragments were used at 1∶5000. BM-purple (Roche) was used as a chromagen to localize hybridized probe. Nuclear fast red was used as a counterstain.Paraffin section in situ hybridization on human fetal tissue was performed as described 5′-GGGCCACCTCCTATGAAAAT-3′; Gcm2R, 5′-GCAGCCTCTAGGGATGTGAA-3′. NBT/BCIP (Roche) was used to localize the hybidized probe. Embryos were embedded in paraffin and sectioned after staining in whole mount.Whole-mount +/Cre;R26-YFP+/tgFoxn1 mice were dissected, minced into small pieces and agitated in RPMI1640 with 2% FBS to remove most thymocytes. The remaining tissue pieces were collected and resuspended in RPMI1640 and 0.2 mg/ml collagenase for 20 minutes at 37°C with gentle stirring. The tissue pieces were allowed to settle for 5 minutes, the supernatant was discarded, and the tissue was resuspended in dispase media  for 20 minutes at 37°C with gentle stirring. The supernatant was discarded and the tissue chunks resuspended in fresh dispase media for 30–45 minutes at 37°C. The digested products were then passed through a 25 G needle, centrifuged at 800× g for 3minutes, resuspended in PBS containing 2% FBS and 5 mM EDTA, and then filtered through a 70 um cell strainer. The filtered cells were stained with anti-mouse CD45-PE (BD pharmingen) antibody before being subjected to sorting using a MoFlo cell sorter (Dako) to isolate PE-, YFP+ TECs. The yield of TECs was about 20,000 cells per adult thymus, with about 93% purity.Thymic stromal cell isolation was modified from a previously described method Pth FAM primer-probe (Assay ID: Mm00451600-g1) was purchased from Applied Biosystems. PCR was performed at 50°C, 2 min; 95°C, 10 min; 40 cycles of 95°C for 15 sec; 60°C for 1 min. The relative quantity of gene expression was determined using 7500 SDS software (Applied Biosystems).Total RNA from sorted TECs was extracted with the RNeasy Micro kit according to manufacturer's instructions (QIAGEN). Total RNA from whole thymi was isolated with Trizol (Invitrogen). First-strand cDNA was reverse transcribed using superscript III (Invitrogen). Quantitative PCR was performed on an ABI 7500 real time PCR system with Taqman universal PCR mix (Applied Biosystems). 18S rRNA VIC/TAMRA primer-probe (Applied Biosystems) was used as endogenous control. Serum sample collection from E18.5 fetal, newborn or adult mice has been described Neonatal thymecotomy was performed as described


This study investigates the impact of tubewell user density on cholera and shigellosis events in Matlab, Bangladesh between 2002 and 2004. Household-level demographic, health, and water infrastructure data were incorporated into a local geographic information systems (GIS) database. Geographically-weighted regression (GWR) models were constructed to identify spatial variation of relationships across the study area. Zero-inflated negative binomial regression models were run to simultaneously measure the likelihood of increased magnitude of disease events and the likelihood of zero cholera or shigellosis events. The aim of this study was to examine the effect of tubewell density on both the occurrence of diarrheal disease and the magnitude of diarrheal disease incidence.In Matlab, households with greater tubewell density were more likely to report zero cholera or shigellosis events. Results for both cholera and shigellosis GWR models suggest that tubewell density effects are spatially stationary and the use of non-spatial statistical methods is appropriate.Increasing the amount of drinking water available to households through increased density of tubewells contributed to lower reports of cholera and shigellosis events in rural Bangladesh. Our findings demonstrate the importance of tubewell installation and access to groundwater in reducing diarrheal disease events in the developing world. Matlab, Bangladesh is a rural area located approximately 50 kilometers southeast of the capital city, Dhaka. Diarrheal diseases are endemic in Matlab and across Bangladesh. This is due to a number of factors, including natural aquatic environments of diarrheal causative agents, high population density, low socioeconomic status, and limited access to clean water. The installation of tubewells is a primary method for decreasing diarrheal disease incidence, giving Bangladeshis an alternative to drinking contaminated surface water. These tubewells draw water several hundred feet from the underlying aquifer to the surface by hand pumps. A sanitary seal, typically of concrete, ideally prevents seepage of ground water into the tubewell. Despite their sealed nature, tubewells may still harbor microorganisms, such as fecal bacteria, as a result of their proximity to latrines or contaminated surface water bodies .Despite such possible contamination of tubewell water, previous studies of diarrheal disease and drinking water interactions have suggested that it is not simply the use of alternative groundwater sources that provides protection against infection, but also the quantity and accessibility -7. Put sVibrio cholerae bacteria; the infective dose is 10,000 or greater. Shigellosis is caused by ingesting a much smaller dose of Shigella bacteria; sometimes as few as ten bacteria are necessary to cause an infection. The primary symptom of cholera is watery diarrhea, while the primary symptom of shigellosis is bloody diarrhea (dysentery). Cholera bacteria naturally occur in the brackish waters of Bangladesh, while Shigella bacteria are linked to human waste contamination of water supplies. We hypothesized that, despite these described differences between the two diseases, tubewell user density would be predictive of diarrheal events for both.Cholera and shigellosis are two of the most commonly experienced diarrheal diseases in Matlab. Cholera is a disease caused by ingesting a large number of baris, or patrilineally related household clusters, which are then further grouped within 142 villages  has conducted continuous demographic surveillance system (DSS) of Matlab's residents. Each resident is assigned a unique identification number which is used to track their birth, place of residence, migration, health events, marriage, and eventually, death. The 225,000 residents of Matlab live in a network of ICDDR,B operates a hospital in southwest Matlab that specializes in high-quality treatment of diarrheal diseases, respiratory infections, and maternal and child health. Treatment at the ICDDR,B is free for residents of Matlab, and free transportation to the hospital is available for residents who cannot afford travel. The laboratory diagnoses and treatment outcomes of patients are linked to their demographic and health records via the resident's identification number.bari are geocoded in the GIS, as are the coordinates of other geographic features, such as the ICDDR,B hospital. The location of features in the Matlab GIS are accurate within 10 meters [The health and demographic surveillance data collected by ICDDR,B is linked to the spatial locations of residence via a Geographic Information System (GIS). The latitude and longitude coordinates collected at the center of each 0 meters . The spabari of ownership. For each bari we calculated the total number of available tubewells  survey of tubewells conducted in Matlab in 2003 with hand-held receivers used for previous surveys . This suvenience . The disbari for the period January 1, 2002 to December 31, 2004. These years were chosen to extend one year before and one year following the tubewell GPS survey in 2003, to avoid misrepresenting tubewell density prior to the survey, and to avoid confounding by the mass installation of deep tubewells by non-governmental organizations (NGOs) in Matlab in 2005. From 2002 to 2004 there were 645 cholera cases and 527 shigellosis cases in Matlab  models can be used to generate hypotheses about non-stationary relationships as well as pinpoint locations that should be subject to more intensive research ,15.t-values. Local regression models are estimated by constructing a spatial weighting matrix and estimating a local weighted regression for each bari and its defined neighbors. Observations are weighted based on proximity to each location i such that nearer points are assigned greater weight than observations further away, based upon the geographic assumption of spatial autocorrelation. A regression estimate is calculated at each observation, including R2 and other goodness-of-fit measures [t-statistics of significance. The local parameter estimates are specific to each location in both logistic and Gaussian GWR and can be displayed graphically using GIS software, indicating potential spatially-heterogeneous patterns in the relationships between diarrheal disease incidence and variables of interest.GWR produces two types of models: a global regression model that treats relationships as spatially stationary, and local regression models estimated at each observation point. Similar to traditional regression models, GWR estimates a model of global associations with parameter estimates, standard errors and measures . A Montebaris were aggregated into 666 clusters using a 25 × 25 meter grid in ArcGIS software. The grid was constructed to create a smooth, continuous surface where each cell has unique coordinates that can be analyzed using GWR. While each bari in Matlab is associated with a specific set of geographic coordinates collected at the center of the bari, the coordinates represent points rather than polygons, their dense spatial configuration made conducting GWR without construction of the 25 × 25 meter grid impossible. Model specifications include a logistic distribution in the absence of a negative binomial option to account for low incidence values and overdispersion, and an adaptive spatial kernel function to assign weights for each local regression estimate. The kernel bandwidth was determined by corrected Akaike Information Criterion (AICc) minimization using all data. Adaptive kernel sample size limits were between 166 and 666 bari clusters. We selected a Monte Carlo significance testing procedure for the local parameter estimates. The four variables calculated in the GIS were then analyzed against the aggregated counts of cholera and shigellosis cases reported to ICDDR,B: number of tubewells per bari, total bari population, distance to the Matlab hospital, and the mean tubewell depth per bari. Specifically, the aim of constructing geographically weighted regression models was to determine whether tubewell density effects on diarrheal events vary across space, when controlling for other covariates. Local variation would suggest that tubewell access intervention strategies are effective in specific areas within Matlab while a lack of spatial variation would indicate equal effects of tubewell access throughout Matlab.Prior to constructing GWR models, the 8732 baris in Matlab from 2002 to 2004, 8278 (94.8%) reported zero cholera events and 8308 (95.1%) reported zero shigellosis events. A zero inflated model assumes that these zero counts are made up of both true and false zeroes. In the case of the Matlab dataset, false zeroes would arise from diseased individuals not seeking treatment at the central Matlab ICDDR,B hospital, either because of self-treatment with oral rehydration or other methods, or because medical attention was sought elsewhere in Matlab or the surrounding region. We assume that these false zeroes are greatly outnumbered by true zero counts, however, because of the Matlab ICDDR,B hospital's excellent record in treating diarrheal diseases, with free treatment options and provision of transport to sick individuals.Many disease datasets contain a large number of zeroes, violating standard regression distribution assumptions and making Poisson or negative binomial regression problematic -19. Zerobari experiencing increased magnitude of disease events (the count model), and the likelihood of a bari experiencing no cholera or shigellosis events (the binary model) [Zero inflated models are comprised of two components which are analyzed separately. The first component is a count model, containing all positive count observations and a proportion of zero counts that can be normally expected under either a Poisson or negative binomial distribution. The second is a binary model, where zero counts are compared to observations with any positive counts. Zero inflated models thus allow us to estimate the effects of multiple variables on both the likelihood of a y model) ,22,23.pscl package in R was used for parameter estimation [The timation ,24,25. Abari population and average hospital distance are significant predictors of a bari cluster reporting a cholera event. The likelihood of a bari cluster reporting a cholera event increases as population increases. Baris located further from the hospital experience a distance decay effect and are less likely to report cholera. Controls for mean tubewell depth and number of tubewells per bari are not significant in the global cholera model.Results for the GWR cholera model can be seen in Tables bari. Parameter estimates are not significant but are shown to demonstrate the limited variability among the relationship between reported cholera and tubewell density across the study area, as indicated by very small range of parameter estimates (between 0.022 and -0.059).All variables included in the global model were tested in the local cholera model. Decreased AIC values suggest that the local model is a better fit for the data as relationships are non-stationary. A decrease in AIC of 3 points or greater is considered evidence of an improved fit . For thebari population is a significant positive predictor of reported shigellosis. Average hospital distance is a significant negative predictor of shigellosis, thus the likelihood of a bari cluster reporting a case of shigellosis between 2002 to 2004 decreases as distance to hospital increases. Mean tubewell depth and number of tubewells per bari variables are not significant predictors in the global shigellosis model.The global GWR results for the shigellosis model are similar to the cholera model results Table . Total bbari and shigellosis are shown in Figure The decrease in corrected AIC between the local and global shigellosis models is less than 3 points suggesting that the local model is not a better fit and spatial heterogeneity is not present Table . Using tResults for the ZINB model for cholera events are presented in Table baris experiencing cholera in 2002 to 2004, there was a strongly significant (p < .001) relationship to population and hospital distance. Baris with larger populations during the time period experienced greater numbers of cholera cases. Baris that were far away from the Matlab ICDDR,B hospital experienced lower cholera counts. The number of tubewells that a bari had access to was not a statistically significant predictor of cholera counts nor was the mean tubewell depth. The binary model for cholera shows only two statistically significant relationships in the prediction of zero cholera counts: number of tubewells per bari and the background population. Increased access to tubewells, as measured by the number of tubewells per bari, significantly increases the probability of a bari reporting zero cholera events. The background population of a bari shows a statistically significant negative relationship with zero counts, such that higher bari population decreases the chances of a bari reporting no cholera. Mean tubewell depth, as in the count model, showed no statistically significant association in the binary model.For bari population from 2002-2004 is a significant (p < .001) positive predictor of positive shigellosis counts: as population increases so does the number of shigellosis cases experienced by a bari. Distance to the ICDDR,B hospital has the opposite effect on shigellosis case counts: baris located further from the hospital are less likely to have high numbers of reported shigellosis cases (p < .001). Neither the number of tubewells in a bari nor a bari's mean tubewell depth are significant predictors of shigellosis counts.As seen in Table bari was able to access and the population of a bari from 2002-2004. As the number of tubewells in a bari increases, the likelihood of reporting zero shigellosis events increases. As population increases, the reporting of zero shigellosis events decreases at the bari level. Distance to the hospital and mean tubewell depth were not significant predictors of whether a bari experienced shigellosis during 2002 to 2004.Only two variables were significant in the shigellosis binary model: the number of tubewells that a Geographically weighted regression models were constructed to explore spatial heterogeneity of relationships to determine whether a stationary statistical model was appropriate for the data. Results from both the cholera and shigellosis models suggest that relationships are stationary. The influence of tubewell density on both cholera and shigellosis counts does not vary across the study area suggesting that water access interventions should apply to the whole of Matlab rather than specific target areas.bari is able to access is an important predictor of diarrheal disease events. The models control for background bari population size, so regardless of the number of people sharing tubewells, as access to tubewell water increases at the bari level the chances of those baris reporting no cholera or shigellosis events also increases. This relationship holds true despite the presence of a mean tubewell depth variable in the model, which would control for the effect of varying tubewell depths in some baris, or the absence of tubewells completely in one third of the baris measured. It is commonly assumed that deeper tubewells draw less polluted water to the surface than shallow tubewells, which may become contaminated as surface water or other material seeps downwards towards the water table [baris without tubewells or in baris with shallower tubewells is accounted for, the total access to tubewells in a bari is a positive predictor for the absence of diarrheal disease.Results from both the cholera and shigellosis zero inflated models indicate that, when controlling for other population and environment covariates, the number of tubewells that a er table -29. Whilbari and the Matlab hospital is a significant variable for both cholera and shigellosis in the count models, wherein baris further from the hospital report fewer cases of disease. We interpret these results to mean that there is a distance decay effect in Matlab, such that even in baris with disease events, fewer are reported to the hospital. This could be due to the long travel times between baris located in the further reaches of the study area, or due to residents seeking medical treatment outside the bounds of the study area. Distance to the hospital is not, however, a significant variable in either of the binary models, suggesting that it is not strongly predictive of whether a bari does or does not experience disease. The global GWR models support this assertion.The distance between a baris with no safe tubewells have shown a variety of strategies, ranging from travelling to neighboring baris or public tubewells to access safe water, switching usage to only one tubewell in a bari, or ignoring the warnings and still using arsenic infected tubewells [Our findings demonstrate the importance of access to clean water in driving diarrheal disease events in the developing world: higher densities of tubewells contribute to lower reports of cholera and shigellosis events. Increasing the amount and quality of clean water available to residents of Matlab and other regions of Bangladesh could lower not only the risk of cholera and shigellosis, but also other diarrheal diseases. This study was limited to the reporting of disease events from 2002 to 2004. Since that time, a large number of very deep tubewells (> 700 feet) have been installed in Matlab, and elsewhere in Bangladesh, by a variety of NGOs to mitigate the impacts of arsenic poisoning from shallow tubewells. Such installation could have dramatic effects on both the distribution and intensity of diarrheal disease events. As part of arsenic mitigation efforts, tubewells were labeled as safe or unsafe for drinking. Residents of ubewells . Our futThe authors declare that they have no competing interests.MC, VE and ME designed the study, and with JM carried out the statistical analysis and drafted the manuscript. MY and PKS oversaw data design and collected the dataset, and helped revise the manuscript. SG and JW participated in the design of the study and helped to draft the manuscript. All authors read and approved the final manuscript.


The dynamic, decentralized world-wide-web has become an essential part of scientific research and communication. Researchers create thousands of web sites every year to share software, data and services. These valuable resources tend to disappear over time. The problem has been documented in many subject areas. Our goal is to conduct a cross-disciplinary investigation of the problem and test the effectiveness of existing remedies.We accessed 14,489 unique web pages found in the abstracts within Thomson Reuters' Web of Science citation index that were published between 1996 and 2010 and found that the median lifespan of these web pages was 9.3 years with 62% of them being archived. Survival analysis and logistic regression were used to find significant predictors of URL lifespan. The availability of a web page is most dependent on the time it is published and the top-level domain names. Similar statistical analysis revealed biases in current solutions: the Internet Archive favors web pages with fewer layers in the Universal Resource Locator (URL) while WebCite is significantly influenced by the source of publication. We also created a prototype for a process to submit web pages to the archives and increased coverage of our list of scientific webpages in the Internet Archive and WebCite by 22% and 255%, respectively.Our results show that link decay continues to be a problem across different disciplines and that current solutions for static web pages are helping and can be improved. Scholarly Internet resources play an increasingly important role in modern research. We can see this by the increasing number of URLs published in a paper's title or abstract also see. Until nThe problem has been documented in several subject areas, with Table Some solutions have been proposed which attack the problem from different angles. The Internet Archive (IA)  and WebCOther tools, like the Digital Object Identifier (DOI) System  and PersIn this study, we ask two questions: what are the problem's characteristics in scientific literature as a whole and how is it being addressed? To assess progress in combating the problem, we evaluate the effectiveness of the two most prevalent preservation engines: and examine the effectiveness of one prototyped solution. If a URL is published in the abstract, it is assumed that the URL plays a prominent role within that paper, similar to the rationale proposed by Wren .2 of 0.96. Its chances of being archived go up after an initial period of flux  abstracts containing "http" in the title or abstract from the years under study (1996-2010), out of which 17,110 URLs  were extracted and used. We developed Python scripts to access these URLs over a 30-day period. For the period studied, 69% of the published URLs (67% of the unique) were available on the live Internet, the Internet Archive's Wayback Machine had archived 62% (59% unique) of the total and WebCite had 21% (16% unique). Overall, 65% of all URLs (62% unique) were available from one of the two surveyed archival engines. Figure 2 of 0.98 while the latter's increase was 174 papers with an R2 of 0.97.How common are published, scholarly online resources? For WOS, both the percentage of published items which contained a URL as well as their absolute number increased steadily since 1996 as seen in Figure A total of 189 (167 unique) DOI URLs were identified, consisting of 1% of the total, while 9 PURLs (8 unique) were identified. Due to cost, it is lZoological Studies (+16) and the top-level domains org and dk (+8 for both). Though smaller in magnitude than the positive ones, the 3 categorical predictors having the largest negative impact were the journals Computer Physics Communications (-4) and Bioinformatics (-2) as well as the domain kr (-3), though the P values associated with the latter two are more marginal than some of the others (.006 and .02 respectively).In order to shed some light on the underlying phenomena of link rot, a survival regression model was fitted with data from the unique URLs. This model, shown in Table While examining URL survival and archival, it is not only interesting to ask which factors significantly correlate with a URL lasting but also which account for most of the differences. To that end, we fit logistic models for each of the measured outcomes  to help tease out that information. To enhance comparability, a similar list of predictors (differing only in whether the first or last year a URL was published was used) without interaction terms was employed for all 3 methods and unique deviance calculated by dropping each term from the model and measuring the change in residual deviance. Results were then expressed as a percentage of the total uniquely explained deviance and are graphically shown in Figure For live web availability, the most deviance was explained by the last year a URL was published (42%) followed by the domain (26%). That these two predictors are very important agrees with much of the published literature thus far. For the Internet Archive, by far the most important predictor was the URL depth at 45%. Based on this, it stands to reason that the Internet Archive either prefers more popular URLs which happen to be at lower depths or employs an algorithm that prioritizes breadth over depth. Similar to the IA, WC had a single predictor that accounted for much of the explained deviance, with the publishing journal representing 49% of the explained deviance. This may reflect WC's efforts to work with publishers as the model shows one of the announced early adopters, BioMed Central , as haviAnother way to measure the effectiveness of the current solutions to link decay is to look at the number of "saved" URLs, or those missing ones that are available through archival engines. Out of the 31% of URLs (33% of the unique) which were not accessible on the live web, 49% of them (47% of the unique) were available in one of the two engines, with IA having 47% (46% unique) and WC having 7% (6% unique). WC's comparatively lower performance can likely be attributed to a combination of its requirement for human interaction and its still-growing adoption.In order to address the discrepancy, all sites that were still active but not archived were submitted to the engine(s) from which they were missing. Using the information gleaned from probing the sites as well as the archives, URLs missing from one or both of the archives, yet still alive, were submitted programmatically. This included submitting 2,662 to the Wayback Machine as well as 7,477 to WebCite, of which 2,080 and 6,348 were successful, respectively.http://faq.web.archive.org/my-sites-not-archived-how-can-i-add-it/) necessitated trusting a message shown by the Wayback Machine when one finds a URL that isn't archived and clicks the "Latest" button. In this instance, the user is sent to the URL "http://liveweb.archive.org/<url>" which has a banner proclaiming that the page "will become part of the permanent archive in the next few months". Interestingly, as witnessed by requests for a web page hosted on a server for which the authors could monitor the logs, only those items requested by the client were downloaded. This meant that if only a page's text were fetched, supporting items such as images and CSS files would not be archived. To archive the supporting items and avoid duplicating work, wget's "--page-requisites" option was used instead of a custom parser.Archiving missing URLs in each of the archival engines had their own special nuances. For the Internet Archive, the lack of a practical documented way of submitting URLs  or the abuse logic. Eventually, we learned that certain URLs would cause WC's crawler to timeout indefinitely, requiring the implementation of a maximum retry count (and a failure status) if the error wasn't caused by the abuse logic.To estimate what impact we had on the archives' coverage of the study URLs, we compared a URL survey done directly prior to our submission process to one done afterwards; a period of about 3.5 months. It was assumed that the contribution due to unrelated processes would not be very large given that there was only a modest increase in coverage, 5% for IA and 1% for WC, over the previous period of just under a year and a half.Each of the two archival engines had interesting behaviors which required gauging successful submission of a URL by whether it was archived as of a subsequent survey rather than using the statuses returned by the engines. For the Internet Archive, it was discovered that an error didn't always indicate failure, as there were 872 URLs for which wget returned an error but which were successfully archived. Conversely, WebCite returned an asynchronous status, such that even in the case of a successful return the URL might fail archival; the case in 955 out of a total of 7,285.Submitting the 2662 URLs to IA took a little less than a day, whereas submitting 7285 to WC took over 2 months. This likely reflects IA's large server capacity, funding and platform maturity due to its age.Converting some of the potential predictors from the list of published URLs to the list of unique URLs presented some unique issues. In particular, while converting those based on the URL itself  were straightforward, those which depended upon a publishing article  were estimated by collating the data from each publishing. Only a small amount, 8%, of the unique URLs, appeared more than once, and among the measured variables that pertained to the publishing there was not a large amount of variety. Amongst repeatedly-published URLs, 43% appeared in only one journal and the presence of funding text was the same 76% of the time. For calculating the number of times a paper was published, multiple appearances of a URL within a given title/abstract were counted as one. Thus, while efforts were made to provide a representative collated value where appropriate, it's expected that different methods would not have produced significantly different results.Even though WOS's index appears to have better quality Optical Character Recognition (OCR) than PubMed, it still has OCR artifacts. To compensate for this, the URL extraction script tried to use some heuristics to detect the most common sources of error and correct them. Some of the biggest sources of error were: randomly inserted spaces in URLs, "similar to" being substituted for the tilde character, periods being replaced with commas and extra punctuation being appended to the URL (sometimes due to the logic added to address the first issue).Likely the largest contributors to false negatives are errors in OCR and the attempts to compensate for them. In assessing the effectiveness of our submissions to IA, it is possible that the estimate could be understated due to URLs that had been submitted but not yet made available within the Wayback Machine.Dynamic websites with interactive content, if only present via an archiving engine, would be a source of false positives, as the person accessing the resource would presumably want to use it as opposed to viewing the design work of its landing page. If a published web site goes away and another installed in its place , then the program will not be able to tell the difference since it will see a valid (though impertinent) web site. In addition, though page contents can change and lose relevance from their original use, dates oAnother source of false positive error would be uncaught OCR artifacts that insert spaces within URLs if it truncated the path but left the correct host intact. The result would be a higher probability that the URL would appear as a higher level index page, which are generally more likely to function than pages at lower levels ,12.Web of Science was chosen because, compared to PubMed, it was more cross-sectional and had better OCR quality based on a small sampling. Many of the other evaluation criteria were similar between PubMed and WOS, as both contain scholarly work and have an interface to download bibliographic data. Interestingly, due to the continued presence of OCR issues in newer articles, it appears that bibliographic information for some journals is not yet passed electronically.2 of .51 and .18 for unique), whereas years  have linear slope 0.031 and R2 .90 (.036 and R2 .95 for unique URLs using the first published year) indicating that the availability between years for older URLs is much more stable whereas the availability for more recent online resources follow a linear trend with a predictable loss rate. Overall, 84% of URLs (82% of the unique) were available in some manner: either via the web, IA or WC.Based on the data gathered in this and other studies, it is apparent that there is still a problem with irretrievable scholarly research on the Internet. We found that roughly 50% of URLs published 11 years prior to the survey (in 2000) are still left standing. Interesting is that the rate of decay for late-published URLs (within the past 11 years) appears to be higher than that for the older ones, lending credence to what Koehler suggested about eventual decay rate stabilization. SurvivaSeveral remedies are available to address different aspects of the link decay problem. For data-based sites that can be archived properly with an engine such as the Internet Archive or WebCite, one remedy is to submit the missing sites which are still alive to the archiving engines. Based on the results of our prototype  mentioned in the introduction, those who administer cited tools could begin to maintain and publish a permanent URL on the web site itself. Perhaps an even more radical step would be for either these existing tools or some new tool to take a Wikipedia approach and allow end-users to update and search a database of permanent URLs. Considering the studies that have shown around at least 30% of dead URLs to be locatable using web search engines , such a For dynamic websites, the current solutions are more technically involved, potentially expensive and less feasible. These include mirroring  and providing access to the source code, both of which require time and effort. Once the source is acquired, it can sometimes take considerable expertise to make use of it as there may be complex libraries or framework configuration, local assumptions hard-coded into the software or it could be written for a different platform . The efforts to have reproducible research, where the underlying logic and data behind the results of a publication are made available to the greater community, have stated many of the same requirements as preserving dynamic websites ,20. InnoThe then-current year 2011) was excluded to eliminate bias from certain journals being indexed sooner than others. For analysis and statistical modeling, the R program  and its 1 was excWherever possible, statistics are presented in 2 forms: one representing the raw list of URLs extracted from abstracts and the other representing a deduplicated set of those URLs. The former is most appropriate when thinking about what a researcher would encounter when trying to use a published URL in an article of interest and also serves as a way to give weight to multiply-published URLs. The latter is more appropriate when contemplating scholarly URLs as a whole or when using statistical models that assume independence between samples.URLs not the goal of this study such as journal promotions and invalid URLs were excluded using computational methods as much as possible in order to minimize subjective bias. The first method, removing 943 (26 unique), looked for identical URLs which comprised a large percentage of a journal's published collection within a given year. Upon manual examination, a decision was then made whether to eliminate them. The second method, which identified 18 invalid URLs , consisted of checking for WebCitation's "UnexpectedXML" error. These URLs were corrupted to the point that they interfered with XML interpretation of the request due either to an error in our parsing or the OCR.http://dx.doi.org". PURL sites were identified by virtue of containing "http://purl." in the URL. Interestingly, 3 PURL servers were identified through this mechanism: http://purl.oclc.org, http://purl.org and http://purl.access.gpo.gov.DOI sites were identified by virtue of containing "To make for results more comparable to prior work as well as easier to interpret analysis, a URL was considered available if it successfully responded to at least 90% of the requests and unavailable if less than that. This method is similar to the method used by Wren, and difSurvival analysis was chosen to analyze living URLs due to its natural fit; like people, URLs have lifetimes and we are interested in discussing them, what causes them to be longer or shorter and by how much. Lifetimes were calculated by assuming URLs were alive each time they were published, which is a potential source of error . Data wasurvreg. In selecting the distribution to use, all of those available were tried, with the logistical showing the best overall fit based on Akaike Information Criterion (AIC) score. Better fits for two of the numeric predictors (number of citations to a publishing paper and number of times a URL was published) were obtained by taking the base 2 logarithm. Collinearity was checked by calculating the variance inflation factor against a logistic regression fit to the web outcome variable. Overall lifetime estimates were made using the survfit function from R's survival library.Parametric survival regression models were constructed using R's To prepare a list of URLs (and their associated data), a collection of bibliographic data was compiled by searching WOS for "http" in the title or abstract, downloading the results (500 at a time), then finally collating them into a single file. A custom program (extract_urls.py in Additional file With the extracted URLs in hand, another custom program (check_urls_web.py in Additional file While investigating some of the failed fetches, a curious thing was noted: there were URLs that would consistently work with a web browser but not with the Python program or other command line downloaders like wget. After some investigation, it was realized that the web server was denying access to unrecognized User Agent strings. In response, the Python program adopted the User Agent of a regular browser and subsequently reduced the number of failed URLs.http://web.archive.org/web/*/" + <the url>. Status was judged by the resulting HTTP status code with 200 meaning success, 404 meaning not archived, 403 signifying a page blocked due to robots.txt and 503 meaning that the server was too busy. Because there were a number of these 503 codes, the script would make up to 4 attempts to access the URL, with increasing back off delays to keep from overloading IA's servers. The end result still contained 18, which were counted as not archived for analysis. For WebCite, the documented API was used. This supports returning XML, a format very suitable to automated parsing [At the end of the live web testing period, a custom program (check_urls_archived.py in Additional file  parsing . For sitThe authors declare that they have no competing interests.JH implemented the tools for data acquisition and statistical analysis as well as performed a literature review and drafting of the paper. SXG implemented an initial prototype and provided valuable feedback at every step of the process, including critical revision of this manuscript.supplement.zip. Contains source code used to perform the study, written in python and R. README.txt contains descriptions for each file.Click here for file


The 14th Sanitation Campaign in 2010 had occurred since the planning, execution and finishing with the participation and institutional support. Through the stimulus coming from the oversight and development of a plan of action organized by the of Infection Control Service involving multiple organizational levels, were put into practice the strategies designed to raise awareness of the importance of hand hygiene.This is an experience in aÂ General Hospital, where among the planned activities were encouraged employee participation in the contest for creating a video simulating the technique of hand washing. The disclosure came through the opening event of the annual campaign that brought the video as a reference made by WHO in the same year. By distributing brochures emphasized the regulation considering an interval of three months for delivery of video and awards. Equipment such as video cameras became available and the evaluation of the videos came about through a jury.Six videos were entered in the contest, had the participation of 42 employees. The videos were presented to members of the Superintendent, SCIH, Marketing and Research and Education. The criteria were: use of the technique of hand washing, use of image, sound, design and evaluation of the material as a means of institutional promotion. After the presentation and selection of the winning video, awards were extended to all employees who participated.Events like this do not replace regular training courses aimed at training of all employees, but call attention to the issue and lead institutional involvement on the basis of a need that is intended to provide safety to the patient against the risk of hospital infection.None declared.


Video-gaming is a common pastime among adolescents, particularly adolescent males in industrialized nations. Despite widespread suggestions that video-gaming negatively affects academic achievement, the evidence is inconclusive. We reanalyzed data from over 192,000 students in 22 countries involved in the 2009 Programme for International Student Assessment (PISA) to estimate the true effect size of frequency of videogame use on adolescent academic achievement in science, mathematics and reading. Contrary to claims that increased video-gaming can impair academic performance, differences in academic performance were negligible across the relative frequencies of videogame use. Videogame use had little impact on adolescent academic achievement. Video-gaming is common among adolescents in industrialized countries, with prevalence rates higher than 75% Presently, evidence on whether video-gaming negatively affects academic achievement is too weak for causal claims Specifically, a number of systematic limitations in the extant literature make it difficult to assess the true relationship between academic performance and video-game use. First, most research has used school grades – which contain an inherent subjectivity on behalf of the assessor, and the effects of assessor expectations on student performance are well-documented  or in collaboration with others (multiplayer). The social aspects of multiplayer games, together with their inherent reward structures, are intended to increase the games' appeal and the time people spend playing One concern with random assignment in videogame research is that, in natural settings, people who experience negative effects from gaming can choose not to play, or may have their gaming behavior curtailed. While research with small samples suggests that boys randomly assigned to be given a new videogame console initially perform poorer academically compared to those who have never owned a games console We reanalyzed one of the largest educational datasets ever produced to estimate the true effect size of video-gaming on academic performance among adolescent gamers, and test for differences between single player and multiplayer gamers.We reanalyzed data from over 192,000 students (aged ∼15 years) across 22 OECD countries assessing the frequency of single- and multiplayer video-gaming , and including standardized psychometric measures of performance in science, mathematics and reading ability As video-gaming is most prevalent in Western industrialized nations, we used three criteria for inclusion in the analyses. To ensure that the country was both Western and industrialised, the country had to be an OECD nation, and be classified by the International Monetary Fund as an advanced economy Science, mathematics and reading ability were all assessed on a scale with an international average of approximately 500 and a standard deviation of approximately 100. For reading assessments, students read a section of text and then answered comprehension questions . For mathematics, students engaged in mathematical calculation and interpretation . For science, students applied scientific thinking  The 2009 PISA dataset contains five sets of plausible values for each of these constructs. These values represent Rasch model estimates of student performance based upon the differences in test version and, thus, a range of plausible performance for each student. In accordance with OECD e represents the residual error term, j represents the value was allowed to vary by school site, and k represents that the value was allowed to vary by country.To examine the relationship between videogame use and academic performance, we first recoded the frequency of single player videogame use into three dummy variables: single player daily , single player weekly  and single player monthly . Thus, someone who never played video games scored three zeros, while a student who played daily scored two zeros and a 1. The same dummy coding was undertaken for the frequency of multiplayer videogame use. Data were analysed using multilevel models and the iterative generalized least squares (IGLS) method ds<0.2). The largest performance decline associated with increased video-gaming was in reading, with a difference that approached, but did not exceed the cut off for a small effect (d = 0.18) between students playing multiplayer games daily and those that never played. All other effects were well below the guidelines for a small effect . Research demonstrates that teachers' assessments of student performance are inherently subjective, and are vulnerable to non-veridical influences, including judgments of the student's attitudes and hobbies An advantage of using multilevel modelling analyses is that the relationship between academic performance and video-gaming is allowed to statistically vary across school sites and countries. Within the present data, there was relatively little variance in the relationship between videogame use and academic performance across countries and schools. However, the fact that some variance occurred, particularly at the school level, suggests that increased video-gaming was associated with reductions in academic performance in some schools, but increased performance in others. Thus, with a view to future research, focusing upon any one school is unlikely to provide a good understanding of video-gaming effects on academic achievement. Our results support the need for psychological and educational researchers to examine effects across appropriately large and diverse datasets to ensure their validity.In sum, across more than 192,000 students in 22 countries, video-gaming behaviour had little effect on psychometrically valid assessments of academic performance in science, mathematics, or reading. The results suggest that the impact of video-gaming on academic performance is too small to be considered problematic.Figure S1Frequency of single-player (top) and multiplayer (bottom) game use and science and mathematics performance including South Korea. Error bars represent 95% confidence intervals. As MLwiN does not calculate confidence intervals for multi-level models, we estimated confidence intervals as 1.96 times the standard error of the multilevel model slopes, as recommended in the MlwiN Manual (TIFF)Click here for additional data file.


Responding to the high burden of cholera in developing countries, the WHO now considers vaccination as a supplement to the provision of safe drinking water and improved sanitation in the strategy for cholera control in endemic settings. Cultural concepts of illness affect many aspects of public health. In the first step of a two-step strategy to examine determinants of cholera vaccine acceptance, this study identified social and cultural features of diarrhoeal illness for cholera control in endemic communities.A cultural epidemiological study with locally adapted vignette-based interviews was conducted in two cholera-endemic communities of Zanzibar. A random sample of unaffected peri-urban (n = 179) and rural (n = 177) adults was interviewed to study community ideas of cholera and shigellosis, considering categories of distress, perceived causes, and help-seeking behaviour.Cholera was recognised by 88%. Symptoms of dehydration were most prominent in reports at the peri-urban site. Interference with work leading to strain on household finances was frequently emphasised. Dirty environment was the most prominent perceived cause, followed by unsafe drinking water and germ-carrying flies. Causes unrelated to the biomedical basis of cholera were reported more often by rural respondents. Rural women had more difficulty (20%) to identify a cause than men . Peri-urban self treatment emphasised rehydration; the rural community preferred herbal treatment and antibiotics. Shigellosis was recognised by 70%. Fewer regarded it as very serious compared with cholera  and regarded it as less likely to be fatal . More respondents could not explain causes of shigellosis (23%) compared with cholera . Community respondents less frequently identified dehydration and contagiousness for shigellosis. Government facilities were preferred healthcare providers for both conditions.This study clarified local views of cholera and shigellosis relevant for diarrhoeal disease control in Zanzibar. The finding that rural women were less likely than men to specify causes of cholera suggests more attention to them is required. Better health education is needed for cholera in rural areas and for shigellosis in general. This study also identified variables for subsequent analysis of social and cultural determinants of cholera vaccine acceptance. Vibrio cholerae O1 or O139. A total of 190,130 cases and 5,143 deaths globally were reported to the World Health Organization (WHO) in 2008 [Cholera is an intestinal disease characterised by acute and profuse watery diarrhoea, caused by the bacterium Cultural concepts about illness and how to treat and prevent it are important for many aspects of public health. The role of various social and cultural factors  has practical implications for behaviour, public health, and disease control that need to be considered. Such factors are also likely to be especially important considerations for the acceptance and demand for vaccines -7. EffecStudies have begun to address questions of vaccine acceptance and demand for diarrhoeal diseases, including recent research on typhoid fever and shigellosis in Asian countries -13, but Shigella spp., is also endemic, and it has a profile of symptoms different from cholera, it was included for comparative study of local experience, meaning and preferred sources of help for diarrhoeal illness.Fieldwork was undertaken in Zanzibar, motivated by the interest of the Ministry of Health and Social Welfare (MoHSW) in using a cholera vaccine for control in endemic peri-urban and rural areas of the archipelago. Because shigellosis, caused by enteropathogenic Specific aims of the study were (i) to examine the variety and distribution of social and cultural views of cholera, (ii) to compare these views in peri-urban and rural endemic communities, and (iii) to identify common and distinctive features of cholera and shigellosis that clarify how well differentiated these conditions are in these communities.The survey was conducted from June to August 2008 in Zanzibar, United Republic of Tanzania. This Indian Ocean archipelago consists of two major islands – Unguja and Pemba – inhabited by a rapidly growing population of approximately 1.2 million Kiswahili-speaking people, who are predominantly Muslim. Medical morbidity in the population of Zanzibar mainly results from communicable diseases like upper respiratory tract infections, including pneumonia , malaria (9.7%) and diarrhoeal diseases (8.6%) . Accordi® was conducted in January and February 2009. Interviews for this study were conducted simultaneously in the peri-urban Shehia of Chumbuni and the rural Shehia of Mwambe. A description of the study sites is given in Table A peri-urban and a rural community  in core areas for a subsequent mass vaccination campaign were selected as study sites. This campaign with the killed whole-cell oral cholera vaccine DukoralAmong the various formulations of cultural epidemiology for health social science research , this stA first version of the interview was drafted in English during several scientific workshops and translated locally into Kiswahili. A series of focus group discussions and a field assistant training workshop with piloting of the instrument among people living adjacent to the study communities followed. This was crucial to further refine the EMIC interview with regard to clarity, field applicability and questions concerning translation. Because people without a current diarrhoeal disease were interviewed, rather than cases, the conditions that were the focus of the interview were introduced as clinical vignettes. For each condition, the respondent was asked to consider the case of a person typical of community residents with pathognomonic somatic symptoms presented in simple, easily understandable terms , perceived causes, self treatment at home, and outside help seeking. The selection of the most relevant locally valid categories of distress, perceived causes, and help-seeking behaviour required for a meaningful description of the insider's perspective was based on discussions with local researchers, fieldworkers and focus group discussions among purposively selected community residents.This cross-sectional survey was conducted prior to a mass oral cholera vaccination campaign to provide baseline data on community views of diarrhoeal illness in areas of Zanzibar at high risk for cholera among unaffected adults . A simplThree field teams plus a coordinator on both islands were recruited by the MoHSW and trained in a ten-day workshop to conduct this survey. Each team consisting of an interviewer and a note taker completed on average two interviews per day. Written informed consent was obtained from all participants prior to the interview and no compensation was offered to them.2 and Fisher's exact test were applied when comparing proportions. This particular approach to comparing prominence, which has been widely used in other cultural epidemiological studies, takes more information about a category into account than a simple comparison of frequencies of report without considering how they are reported.For cholera, the categories related to illness experience, meaning and help-seeking behaviour were coded for their prominence with a value of 2 after a spontaneous response, a value of 1 after a probed response and a value of 0 if not considered at all to reflect the response style. An additional value of 3 was assigned to the category of response if the category was considered the most troubling category of distress, the most important perceived cause or the most helpful self treatment or source of help. The cumulative prominence by respondent (ranging from 0-5) was then used to calculate the mean prominence for each category. Thematically similar individual categories were grouped under specific headings (e.g. related to dehydration among somatic symptoms) for the analysis of broader concepts of experience, meaning and behaviour. Calculation of the grouped prominence followed the same procedure as with the individual variables. To identify significant differences for cholera between the two sites and between sexes, a non-parametric statistic, the Wilcoxon rank-sum test, was used when comparing prominence variables; the Pearson Chi2 test for paired data was used. To examine whether or not individual categories were differentiated between both conditions, Cohen's kappa was calculated. The kappa statistic indicates the strength of agreement for a categorical assessment, corrected for agreement by chance. The analysis identified the two conditions as distinct for a category if the kappa coefficient was below 0.4, a level commonly accepted as a threshold for moderate agreement [A similar series of questions were asked to elicit shigellosis-related illness experience, meaning and help-seeking behaviour. The same categories that were coded for cholera were also coded for shigellosis. Comparative analysis between the two conditions considered only spontaneously reported categories, because the interview coded only spontaneous responses for shigellosis. The proportion of positive responses by category was tabulated individually for each vignette, and for a report in both vignettes. To determine whether a category was associated more with one vignette than the other, McNemar's Chigreement .Narrative information was written down during the interview in Kiswahili, then translated into English and typed in a word processor. The qualitative software MAXQDA, version 2007, was used for managing the textual data and to facilitate further analyses of findings from quantitative data. Quantitative data was entered twice and verified in Epi Info software, version 3.4.3, and cleaned. Statistical analyses were done with Stata, version 10.The sample size calculation was based on comparison of mean prominence of categories of distress, perceived causes, self treatment and outside help seeking for peri-urban–rural and female–male differences. The detection of a difference of 0.5 between prominence means with equal standard deviations of 1.5 at 95% significance and 80% power required a sample size of at least 164 individuals per independent group. This calculation was based on a two-sample t test assuming no underlying distribution in the data . Ten perThe protocol describing the study presented here was cleared by the WHO Research Ethics Review Committee and the MoHSW Ethics Committee in Zanzibar and later published in an open access journal to make it freely available to the research community . Only inA total of 356 interviews were conducted, with very few people among the visited households who refused to be interviewed. The socio-demographic characteristics of the sample are summarised by site in Table kipindupindu, which is the Kiswahili name for the disease entity cholera. The rural villagers recognised cholera less often than the peri-urban residents . Other names given by rural villagers were kuharisha kawaida for normal diarrhoea (6.2%) and kuharisha maji for watery diarrhoea (4.0%) while 6.2% could not identify the condition at all. The condition described in the shigellosis vignette was identified by 69.9% of the respondents as kuharisha damu, which refers to the disease entity bloody diarrhoea. While 12.9% could not name it at all, 19 individuals (5.3%) confused the case presented in the shigellosis vignette with cholera.The vignette describing an adult person with symptoms of acute watery diarrhoea was named by 88.2% of the sample as 2 test). Cholera was also more often anticipated to be "usually fatal without treatment" (77.5%) than shigellosis . Although there was no difference in perceived severity for cholera at the two sites, for shigellosis more peri-urban respondents considered it very serious (86.0%) than rural respondents . Peri-urban respondents more frequently anticipated fatality for cholera (84.4%) than rural respondents , and peri-urban respondents were also more likely to anticipate fatality for shigellosis (65.4%) than rural respondents .The perceived severity and likely fatality for cholera and shigellosis vignettes was assessed in the peri-urban and rural areas. Cholera was more frequently said to be "very serious" (96.6%) than shigellosis , but not between women and men .When asked about previous experiences of the condition described in the cholera vignette, 5.3% of the total sample reported an individual episode. Stratified analyses revealed a significant difference between the peri-urban and rural community , related to general in-and outdoor dirtiness, was by far the most prominently reported perceived cause overall, but particularly notable in the peri-urban site "It is possible that the flies coming from the toilet contaminate the food." Among the causes not related to the faecal-oral route of transmission, God's will was the most prominent category and ranking higher among rural residents. A statement from a 30-year-old female farmer from Mwambe helps to explain the commonly expressed notion regarding this finding, i.e. that God overrules people's prevention efforts if only it wished:"There is no cause except God's will, which cannot be changed; and it is not caused by dirty environment because there are some dirty places where people do not get the disease."Further perceived causes not linked to cholera disease aetiology – like witchcraft, malaria and worms – had lower prominence ratings since they were almost never mentioned spontaneously nor identified as most important. And these categories were more characteristic for the rural compared with the peri-urban community. A substantial proportion of the respondents from Mwambe – more than one-tenth, compared to only two peri-urban residents – had no idea what could have made the person suffer from the symptoms described in the vignette (coded as cannot say). Among rural respondents who could not spontaneously identify a cause, women featured significantly more often than men  (not shown in Table mpatakuva (Plectranthus spp.), neem tree, guava). Doing nothing at home, i.e. sending the person described in the cholera vignette immediately to allopathic healthcare facilities, was considered as the least prominent category in the rural community, while it ranked fourth in the peri-urban community and was regarded as the most helpful thing one can do at home. The following statement from a housewife, aged 47 years, from Chumbuni is typical for what the communities would do for people with cholera at home:The most prominent self treatment at home in the rural community was herbal treatment, followed by giving antibiotics or other drugs like pain killers or antacids and then home-made or ready-to-use oral rehydration solution (ORS) .Findings from both peri-urban and rural areas of Zanzibar were notable for the high perceived severity and anticipated fatality of cholera. Even though the condition described in the cholera vignette was similarly regarded as very serious in both communities, it was more often named as cholera and considered as a serious life-threatening illness in the peri-urban community. The lower recognition of the condition described in the cholera vignette in the rural community, which is consistent with lower prominence of reported signs and symptoms of dehydration and higher prominence for the two most conspicuous shigellosis signs , may be explained by poorer education. It cannot be explained by less personal illness experience of cholera, however, since rural residents reported the occurrence of an individual episode 2.8 times more often than peri-urban residents.The severity of the condition in the cholera vignette was also elaborated with reference to its impact on affected persons and household livelihoods. Absence from work was felt to be the major effect at both sites leading to strain for household finances because of reduced or lost income and treatment costs. Compared to the shigellosis vignette, the condition described in the cholera vignette was more often perceived as a severe and potentially fatal health problem in both communities. This finding is consistent with another study comparing the two conditions; unaffected community residents, confirmed shigellosis patients and healthcare providers in Bangladesh considered cholera to be more severe than shigellosis .Although a variety of causes were acknowledged, respondents clearly regarded the condition depicted in the cholera vignette as a disease linked to a dirty environment and to ingesting microbiologically contaminated water and food. The relevance of this concept of dirtiness and of sanitation and hygiene in connection with diarrhoea was also found in a qualitative study of childhood diarrhoea among mothers living in Chake-Chake district on Pemba . The rolBesides using various allopathic and traditional home remedies, respondents also recommended immediate hospital treatment when queried about what they would do at home with someone like the person described in the cholera vignette. Peri-urban community responses emphasised rehydration; rural community responses emphasised herbal treatment and use of antibiotics and other drugs. Certain herbs and plants, most of which were also reported as herbal treatment in the childhood diarrhoea study from Chake-Chake , were frIn both sites, help seeking outside the household for the person described in the cholera vignette essentially meant going to public healthcare facilities, with little mention of traditional healers and faith healers. This finding of reliance on hospital treatment is remarkable compared with other studies from low-and middle-income countries, which emphasise traditional treatment for childhood and adult diarrhoea -33. SeveThe analysis of disagreement showed illness concepts for the two conditions were distinct with respect to reported patterns of distress, perceived causes and self treatment. For outside help seeking, however, reference to the value of hospital treatment was the same for both conditions. Differentiation of the two conditions may be explained by community and personal experience with cholera and shigellosis, resulting in the awareness of particular features of the two conditions. Both conditions occur with similar rates in the study communities Table .Health educational activities for cholera, in response to the priority arising from outbreaks making heavy demands on the health system in Zanzibar, are more extensive than for shigellosis. Less emphasis on shigellosis control may account for the finding that fewer respondents could explain the cause of shigellosis (23% reporting cannot say) compared with cholera (7.3%). The finding that fewer respondents identified houseflies as a cause of shigellosis may also result from the lower priority of public health action for shigellosis control, inasmuch as houseflies are recognised agents of transmission for shigellosis . DehydraThe differentiation of the two conditions is reflected by appropriate differences in treatment recommended by respondents. Community self-treatment priorities emphasised rehydration for cholera and herbal and antibiotic treatment for shigellosis.This study shows how EMIC interviews can be used to assess explanatory models of diarrhoeal illnesses among unaffected community residents and how to compare them among sub-groups. The specific approach employed in this cultural epidemiological study to comparing prominence allowed the ranking of categories according to their relative priority and not just according to their reported frequency. This weighted approach represents a more sensitive method to clarify differences between groups and has implications for explaining cultural priorities and potential effects on health behaviour.The findings presented here are specific for cholera and shigellosis in one culture and focus on variation between peri-urban and rural areas. Thus, any generalisations made to countries outside the target populations have to be examined cautiously as the results presented here are inherently linked to the context. Some may argue that the differences in community views are due to education rather than to residence. However, analysis of the patterns of distress, perceived causes, self treatment and help seeking, stratified by educational status, showed that the cross-site differences reported here were not confounded by education.It should also be noted that findings reported here are cross-sectional and may change over time, possibly in response to access to health services, a vaccine campaign or other social changes. Furthermore, the data are based on respondents' ideas about the condition of a clinical vignette, representing community views of illness experience, meaning and behaviour, but not necessarily an account of personal or family history.The sampling included only community residents who were at home when the field teams visited. The study could be biased if the views of the respondents available for interviews at home and persons unavailable because of other responsibilities differed. The age distribution at both sites, however, mitigates this concern, inasmuch as all age groups were represented in the sample.This study has clarified local peri-urban and rural views of cholera among the general population with practical significance for cholera control in Zanzibar. Cholera was recognised as a serious and potentially fatal condition, a priority that makes such communities receptive to community health education programmes. The overwhelming preference for public healthcare facilities to treat cholera and shigellosis indicates the importance of strengthening health systems to ensure they are capable of fulfilling expectations. Notwithstanding this appropriate community preference for hospital treatment, this study also suggests that better health education is needed for cholera in rural areas and for shigellosis in general. The finding that rural women were more likely than men to be unable to specify a cause of cholera indicates the need to ensure a gender-sensitive approach to control.Although sanitation, hygiene and safe water are critical issues for diarrhoeal disease control, recent consideration of vaccines in endemic areas suggest an appealing complementary intervention. It is an approach that has been of considerable interest to policy makers in Zanzibar, where a cholera vaccine campaign was implemented in January and February 2009. Research is needed to identify not only health system capacities to deliver vaccines but also social and cultural factors affecting community acceptance of vaccines. Factors influencing the willingness and enthusiasm of communities for a recommended vaccine can be expected to affect the success of a vaccine intervention programme. The interests and findings of this study are likely to inform such efforts to clarify social and cultural features of vaccine acceptance and demand.Although not used in planning the cholera vaccine campaign in Zanzibar, findings from this study identified variables for a subsequent analysis of social and cultural determinants of vaccine acceptance and demand. Further analysis is also needed to explain the impact of the vaccine campaign on community views of cholera and risk-related behaviour. This study indicates directions and enables further research, and it has also clarified important issues for cholera control.The authors declare that they have no competing interests.Conception and design of this study: CS, AMK, SMA, RH, CLC and MGW. Supervision of data collection and data entry: CS. Analysis of data: CS and MGW. Writing of manuscript: CS and MGW. Revision and final approval of manuscript: CS, AMK, SMA, RH, CLC and MGW.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/10/339/prepubClinical vignettes for community study of cholera and shigellosis.Click here for file