Molecular studies document the prevalence of these mutations in parasite populations across the African continent. However, there is no systematic review examining the collective epidemiological significance of these studies. This meta-analysis attempts to: 1) summarize genotype frequency data that are critical for molecular surveillance of anti-folate resistance and 2) identify the specific challenges facing the development of future molecular databases.Mutations in the dihydrofolate reductase  genotype and dhps double mutant (A437G and K540E) genotype in Africa. A hierarchical mixed effects logistic regression was used to examine the influence of various factors on reported mutant genotype frequency. These factors include: year and location of study, age and clinical status of sampled population, and reporting conventions for mixed genotype data.This review consists of 220 studies published prior to 2009 that report the frequency of select dhfr and dhps mutant genotype frequencies from all African studies that met selection criteria was created for this analysis. The map illustrates particularly high prevalence of both the dhfr triple and dhps double mutant genotypes along the Kenya-Tanzania border and Malawi. The regression model shows a statistically significant increase in the prevalence of both the dhfr triple and dhps double mutant genotypes in Africa.A database consisting of dhfr triple mutant and dhps double mutant genotypes in Africa are consistent with the loss of efficacy of SP for treatment of clinical malaria in most parts of this continent. Continued assessment of the effectiveness of SP for the treatment of clinical malaria and intermittent preventive treatment in pregnancy is needed. The creation of a centralized resistance data network, such as the one proposed by the WorldWide Antimalarial Resistance Network (WWARN), will become a valuable resource for planning timely actions to combat drug resistant malaria.Increasing prevalence of the  Global malaria control initiatives have a turbulent history driven by a number of biological, social, and economic factors. The Malaria Eradication Programme launched by the World Health Organization (WHO) in 1955 successfully eliminated malaria in many temperate and sub-tropical regions of the world, but little progress was made in sub-Saharan Africa. The programme confronted technical challenges including, but not limited to: anti-malarial drug resistance, insecticide resistance, lack of infrastructure to implement and sustain control measures, and a lack of community engagement and participation .The renewed vision for malaria control and eventual elimination focuses on the development of new tools and improved integration of existing interventions . FinanciP. falciparum became a major challenge for control programs [ACT is a vital component of the malaria control strategy and currently represents the last line of defense against infections that have developed resistance to previous anti-malarial therapies. Chloroquine (CQ) monotherapy was highly effective in treating malaria for several years, but eventual appearance of CQ resistance to programs -8. The oprograms ,10.Drug resistance continues to present a major threat to remaining effective anti-malarial therapies, such as ACT, that currently serve as a cornerstone of the malaria control strategy. Therefore, the success of control efforts relies heavily upon surveillance methods to adequately monitor and promptly respond to emerging resistance.Clinical efficacy trials that measure the therapeutic failure rate following administration of a particular drug are the gold-standard method for gauging the effectiveness of anti-malarial therapies. However, clinical efficacy trials are resource-intensive, making them difficult to use broadly for surveillance purposes, reviewed in . Translain vitro-based assays have been used as tools to examine the role of host-drug and parasite-drug interactions that influence clinical efficacy of anti-malarials. Specifically, pharmacological studies conducted within the context of clinical trials can enable an examination of pharmacokinetics and pharmacodynamics (PK-PD) and their role in therapeutic response. An understanding of PK-PD in different clinical settings and populations is essential to establish therapeutic drug dosages for target groups and distinguish between true therapeutic failures and other factors such as poor adherence, drug quality, absorbance, and elimination. PK-PD data is also essential if outcomes from different clinical efficacy trials are to be accurately compared.Pharmacological and In vitro parasite susceptibility assays enable a measure of intrinsic chemosensitivity of P. falciparum parasites to a particular drug by controlling for host factors such as acquired immunity, nutritional status, and compliance dosing. Most in vitro studies report the 50% inhibition concentration (IC50) of an anti-malarial drug, which in turn serves as an indicator of parasite tolerance to the drug. In vitro assays offer benefits, such as the ability to quantify the parasite inhibiting properties of partner drugs within combination therapies and identify temporal trends by comparing the drug sensitivity profiles of frozen reference parasite lines with samples in question. Both in vitro and pharmacological studies play an important role in differentiating drug resistance from other potential causes of decreased drug efficacy. Although these methods complement clinical efficacy trials to collectively identify the presence of drug resistance and help quantify its impact on drug efficacy, they do little to propose specific parasite resistance mechanisms.in vitro and pharmacological studies in that they provide the means to identify genetic markers that confer resistance. Association and genetic complementation studies have identified and validated genetic markers of resistance for older anti-malarial drugs, such as CQ, SP and chlorproguanil-dapsone [Molecular studies examining genetic correlates of resistance differ in scope from -dapsone -24. Mole-dapsone . Lessonsin vitro, and molecular studies each shed light on a certain aspect of drug efficacy and drug resistance, but each also has its own set of benefits and limitations. Lack of standardization across studies of each type and differences in conventions for reporting data are two pervasive problems that seriously limit the ability to compare studies conducted at different times and locations. In an effort to mitigate these and other issues, the WorldWide Anti-malarial Resistance Network (WWARN) has put forth a comprehensive plan to network clinical, in vitro, pharmacological, and molecular studies in a centralized database. The proposed structure and objectives for each of the four components of the database have been detailed in a series of articles [Clinical, pharmacological, articles ,26-28.et al [The need to standardize and network molecular data is particularly pressing. The advancement of high-throughput molecular techniques has enabled a rapid increase in the number of molecular studies conducted in recent years; however, a lack of meaningful ways to compare studies severely limits the potential utility of this valuable information. Databases that assemble the reported frequency of resistant parasite genotypes from multiple molecular studies can provide more comprehensive and easily accessible information. Drug resistance maps at the London School of Tropical Medicine and Hygiene  has asseet al  used a mA growth in the number of molecular studies reporting the frequency of known resistance markers presents new opportunities to assess the broader impact that reporting conventions and study methodology may be having on the data being produced. Some of the variables that have been proposed as potentially influential include age and clinical status of the sampled population, date and location of the molecular study, reviewed in , and repResistance to anti-malarial drugs for which validated molecular markers are available is already widespread; however, a database that specifically assembles studies reporting the frequency of parasite genotypes associated with anti-folate resistance is valuable for a number of reasons.® (CTX), is a low cost regimen that has been shown to decrease morbidity and mortality in HIV-infected individuals [P. falciparum genotypes following CTX treatment [in vitro [in vivo [First, SP use in combination with artemisinin-based derivatives ,34 and aividuals . Howeverividuals . Thus, treatment , continuin vitro  and has [in vivo ,43, even[in vivo ,45. MoniPlasmodium parasite life cycle [The anti-folate class of drugs consists of compounds that bind enzymes necessary for parasite folate biosynthesis. The most widely used anti-malarial drugs within this class are sulphadoxine-pyrimethamine (SP) and, more recently, chlorproguanil-dapsone. The pyrimethamine portion of SP and chlorcycloguanil, the active metabolite of chlorproguanil, bind the enzyme dihydrofolate reductase (DHFR) . Sulphadfe cycle .dhfr and dhps genes of P. falciparum parasites have been associated with decreased parasite sensitivity to the anti-folate drugs. A change from wildtype Ser108 to Asn108 (S108N) in dhfr is sufficient to cause low level pyrimethamine resistance both in vitro and in vivo [in vitro resistance to pyrimethamine by 100-fold relative to wild-type [dhfr can yield higher levels of SP resistance in vitro and in vivo [dhfr gene have evolved in different parts of the world  in combination with the dhfr triple mutant genotype  is a better predictor of clinical failure than either multiple mutant genotype alone [Mutations in codons Ser436 to Ala or Phe (S436A/F), Ala437 to Gly (A437G), Lys540 to Glu (K540E), Ala581 to Gly (A581G), and Ala613 to Ser or Thr (A613S/T) in  dapsone ,59,60. T in dhps . Multiplendently . Howeverpe alone .dhfr and dhps mutant genotypes in African P. falciparum populations; 2) highlight the significance and limitations of these studies; 3) use condensed tables, plots, and geostatistical illustrations to present frequency data; 4) use statistical models to describe changes in the prevalence of select highly resistant genotypes over the years; and 5) elucidate the impact of variables that may influence reported genotype frequencies.The objective of this meta-analysis are to 1) provide a comprehensive database of reported frequencies for resistant dhps," "dihydrofolate reductase," "dhfr," "Plasmodium falciparum," "Africa," "sulphadoxine pyrimethamine," "antifolate," "drug resistance," "Fansidar." Boolean operators were used to search for articles using these terms in various combinations. All variations in the spelling of these terms were accounted for in the search. Additional articles were found through the Related Articles search feature on the Pubmed website and through the reference sections of candidate articles. The Drug Resistance Maps database [A literature search was done using the National Library of Medicine Pubmed/Medline search engine. Search terms included "dihydropteroate synthase," "database  was alsost, 2009 to be included.1) The article must have been published in English prior to Jan 1dhfr  and/or dhps  to be included.2) The article must explicitly state or provide interpretable mutation frequencies for any of the selected codons in in vitro cultured blood isolates were also excluded.3) The article must have provided mutant frequency data derived from pre-treatment blood isolates to be included. Genotype frequency data derived from patients receiving anti-malarial drugs within two weeks of enrollment and/or patients who were determined to have therapeutic levels of anti-folate drugs in their bloodstream at the time of enrollment were excluded. Genotype frequency data derived from 4) The study must have restricted its sampling to blood isolates of African origin from local patient populations to be included. Genotype frequency data derived from travelers were excluded.dhfr and dhps were extracted from published articles and were summarized using two separate databases called the Mutant Allele Frequency (MAF) database and Mutant Genotype Frequency (MGF) database  and dhps double mutant genotype  using a standardized system, similar to the format described by Kublin et al [Data reporting the frequency of mutations in in et al . Table 2One of the major methodological differences across studies was the reporting standards for mixed mutant genotypes. Mixed mutant genotypes are detected when two or more populations of parasites with distinct genotypes at particular codons of interest are present in a single infection. These infections are commonly referred to as "polyclonal" since multiple clonal populations of parasites are present in a single blood isolate. Varying classifications for mixed genotypes can have a significant impact on the determination of mutant genotype frequency; particularly in high transmission settings where polyclonal infections have been reported in greater than 50% of sampled isolates, reviewed in .A majority of studies included in this review used the Restriction Fragment Length Polymorphism (RFLP) technique to differentiate between the mutant and wildtype form at codons of interest. Since this is an indirect method to detect codon change it has some inherent limitations in identifying appearance of new mutations and in accurately genotyping mixed infections . A numbedhfr triple mutant genotype and the dhps double mutant genotype  that is generated by the ArcGIS software. The software determines the optimal (p) value for the map by minimizing the root mean square prediction error (RMSPE), which is a summary statistic that describes the error of the prediction surface. A (p) value of "0" means a given data point has equal influence on all parts of the prediction surface regardless of the distance from the point. Whereas, a large (p) value means the influence of a data point drops off very quickly as the distance from the point increases. The optimal (p) value for each map is shown in Figures An inverse distance weighted (IDW) algorithm was used to provide a snapshot of The prediction surface of each map is shaded using a gradient scheme consisting of quintiles. Areas of the prediction surface considered to have a high prevalence of mutant genotypes are denoted by darker shading, whereas areas considered to have a lower estimated mutant genotype prevalence are denoted by lighter shading. The IDW spatial representation does not account for the date of the study. Therefore, shaded areas of the maps can only serve as crude snapshots of mutant allele prevalence between 1993 and 2008 with decreasing reliability as distance from observed data points increases. The MARA  map descet al [Time plots were created , and also helps to avoid convergence issues in fitting the model . Year enThe fixed effect representing the age of sampled population are divided into five age categories: 1) adolescents and adults, 2) children and adolescents, 3) children under 6 years, 4) mixed ages, 5) N/A. For this study, individuals age 12 and under are considered children, between 12 and 18 years of age are considered adolescents, and age 18 or greater are considered adults. A study is assigned to age category 1, 2, or 3 if greater than 75% of the sampled population for that study fit the category description. If the age of the sampled population is described but did not meet the 75% threshold then it is assigned to the mixed ages category. The N/A category is used to describe studies that did not describe the age of the sampled population.The fixed effect representing mixed genotype categorization is divided into four categories based on the methodology used for reporting mixed genotypes: 1) separate, 2) combined, 3) excluded, and 4) N/A. The separate category is assigned to studies that report mixed genotypes separate from pure mutant or pure wildtype genotypes. The combined category is used to describe studies that counted mixed genotypes as pure mutant genotypes. The excluded category is used to describe studies that completely removed mixed genotype data. The N/A category is used to describe studies that did not describe mixed genotypes in the study. The criteria used to categorize studies based on reporting of mixed genotype data is more thoroughly described in Table ij Ybe the number individuals with the mutant genotype in the th jstudy of the th icountry. Conditional on the fixed and random effects, Yij ~ Binomial, where ij nis the number of individuals in the th jstudy of the th icountry. On the logit scale, the probability of mutation ij πis modeled as a linear combination of the fixed and random effects. Equation (1) represents the logistic regression model for dhfr triple mutant genotypes, and equation (2) represents the logistic regression model for the dhps double mutant genotypes.Let i = 1, ..., number of countries; j = 1, ..., number of studies in the th icountryi vis the intercept random effect of the th icountry, ij uis the intercept random effect of the th jstudy in the ith country, and β is a vector representing the fixed study level effects. The study and country random intercepts allow the intercept to differ between studies. For model (1) the country random effects i vare assumed to be normally distributed i v~ N, while the study random effects ij uare assumed to be normally distributed ij ~ Nu. For the analysis of dhfr triple mutant genotypes, σv, dhfr2 represents the heterogeneity between countries, and σu, dhfr2 represents the heterogeneity between studies within a country. In model (2) the study random effects uij are assumed to be normally distributed uij ~ N, and σu, dhps2 represents the heterogeneity between studies of the dhps double mutant genotypes. The variance components are estimated on the logit scale, and therefore do not have a direct interpretation. Statistical analysis was performed in R version 2.8.1 using the lmer function in the lme4 package, which uses Laplace approximation to the likelihood to obtain parameter estimates [p-values less than 0.05 are considered statistically significant.Here, α is the fixed intercept, stimates . The sigstimates ; p-valuedhfr and/or dhps genotypes. Of the remaining articles, several were excluded on the basis of their location outside of Africa. Few studies were excluded on the basis of other criteria . Data was successfully extracted from 100 articles that yielded genotype frequency information for 220 studies conducted in 31 African countries and published prior to 2009. Of these 220 studies, 207 studies reported the frequency of one or more mutations of interest within the dhfr or dhps gene. These mutations include dhfr A16V, C50R, N51I, C59R, S108N/T, I164L and dhps S436A/F, A437G, K540E, A581G, A613 S. Studies reporting the frequency of these individual mutations are summarized in the mutant allele frequency (MAF) database  or the dhps double mutant genotype (A437G and K540E), or both. These data are summarized in the multiple mutation (MM) database  of the dhfr triple mutant genotype in parts of Cameroon, Uganda, Kenya, Tanzania, and Malawi.The map describing the prevalence of the a Figure  shows thdhps double mutant genotype across Africa , displays the variability of the probability of mutation between countries. Having a random intercept near zero implies that the study's country level random intercept in the logistic regression model is close to the overall fixed continent intercept, α. Countries with smaller random country effects are estimated to have a lower probability of mutation at the start of the study time period compared to countries with larger random effects. The study level random effects are nested within the country effects, and therefore are centered at the country level random effects. The study level random effects (denoted by a hollow circle) are displayed as i v+ ij uand show the variability of the probability of mutation between studies within the same country. The y-axis of Figure Multilevel mixed-effects regression was used to assess the association of several variables on the reported frequency of both the dhfr triple mutant genotype and has a positive parameter estimate. Therefore, the model suggests that, conditional on the random effects, the reported frequency of this genotype in Africa is increasing over time. Of the three remaining variables that were examined, only mixed mutant categorization had a significant (P = 0.01) association with the reported frequency of the dhfr triple mutant genotype. The combined, excluded, and N/A categories all had a positive parameter estimates relative to the separate category; however, only the combined and excluded categories significantly differed from the separate category. This suggests that studies reporting mixed mutant genotypes as pure mutants or excluded them completely tend to have significantly higher reported frequency of the dhfr triple mutant genotype than studies that did not describe mixed mutant genotypes. The age of the sampled population and the type of study  were not significantly associated with reported mutant genotype frequency. A summary of model results for the dhfr triple mutant genotype can be found in Table For the fixed effects, the year of the study is significantly associated (P < 0.01) with the frequency of the dhps double mutant genotype  were excluded from the statistical analysis because they were conducted earlier and could be too influential. A data shortage for the dhps double mutant genotype analysis led to convergence issues that did not allow for estimation of a country level random effect. However, the study level effect was still included in the model as a random intercept effect in order to allow for variation in the probability of mutation at the start of the study time period. As in Figure dhps double mutant genotype is highly significant (P < 0.01) and is estimated to be 8.83. This is much larger than the between or within country variation observed in the analysis of the dhfr triple mutant genotype because the variance for the dhps double mutant genotype could not be partitioned. This suggests that there is significant variability in the reported frequency of the dhps double mutant genotype among different studies at the start of the study time period.The model for the dhps double mutant genotype and has a positive parameter estimate. Therefore, the model suggests that, on average, mutant prevalence is increasing over time in Africa. The three remaining variables that were examined; mixed mutant categorization, age of sampled population, and type of study, were not significantly associated with the reported frequency of the dhps double mutant genotype. A summary of model results for the dhps double mutant genotype can be found in Table The year of the study is significantly associated (P <0.01) with the reported frequency of the dhfr and/or dhps genotypes in Africa to be included in this meta-analysis. The studies were conducted as early as 1993 and as late as 2006; however, the distribution is skewed (mean year: ~2001 for studies reporting either multiple mutant genotype) towards recent years reflecting an increase in the number of studies reporting the frequency of the dhfr triple mutant and/or dhps double mutant genotypes. This observation is in line with the increase in the number of anti-malarial clinical efficacy trials reported in the literature in recent years [dhfr triple mutant genotype and nearly 8,000 individuals for the dhps double mutant genotype. The clear emphasis on reporting the dhfr triple mutant genotype is likely due to its emergence some 10-20 years earlier than the dhps double mutant genotype in Africa [dhps double mutant genotype (437G/540E), on the other hand, emerged much later in the early to mid-1990's [Broad inclusion criteria allowed most studies that report the frequency of nt years  and liken Africa  and its d-1990's  and is odhfr triple mutant genotype  are more likely to yield mixed genotypes than parasite populations that have a low diversity for a particular gene. Thus, differences in the way mixed genotypes are reported have a more pronounced effect on the prevalence rates reported for a gene that has greater diversity in a given population than one that does not. It has been shown that the dhps  mutant genotype is either ubiquitously present or absent in most of the populations where it has been examined [dhps double mutant genotypes along with another dhps mutant genotype are less likely to occur. However, the dhfr triple mutant genotype has historically been found alongside a higher prevalence of sensitive, single, and double mutant dhfr genotypes circulating in the population [dhfr genotype became fixed in the population. This phenomenon may explain why reporting conventions for mixed genotype data was significantly associated with the reported frequency of the dhfr triple mutant genotype but not the dhps double mutant genotype.This meta-analysis shows that there are some important differences in the reported frequency of the n Africa , pervasin Africa , and humn Africa , may expexamined ; therefopulation ,88; therin vitro studies, and standardized interpretation of existing mixed genotype data [dhfr triple mutant genotype and dhps double mutant genotype, there are also some similarities - The prevalence of both alleles is increasing on average in Africa. On average, the age and clinical status of the sampled population are not significantly associated with the reported frequency of either mutant genotype. This is in contrast to smaller regional studies that have shown association between age, treatment failure rate, and the prevalence of genetic markers for resistance [The specific reasons responsible for the differential impact of reporting conventions for mixed genotype data on the reported frequency for either mutant genotype examined in this meta-analysis are difficult to identify and may also be an artifact of confounding variables that were not accounted for in the statistical regression used herein. Regardless, the fact remains that polyclonal infections commonly occur in many of the high transmission settings across Africa and the characterization of mixed genotypes is an issue that must be addressed. Novel methods for analyzing mixed infections , multivaype data  have allsistance ,91. The dhfr triple mutant and dhps double mutant genotypes but could not be examined in this analysis. This was due to lack of power from limited datasets and inability to link these data to reported frequency data where this information was not specifically reported.One of the major limitations in this meta-analysis was the small number of variables that could be assessed in the statistical models. There are a number of clinical and epidemiological variables such as transmission, regional drug administration policies, and target populations  that may better explain the reported frequency of the dhfr and dhps multiple mutant genotypes included in this analysis were conducted over a period of 13 years. In most cases, the data from a particular country only spans a fraction of this 13 year period; therefore, there is a regional bias where data from a particular year is really a reflection of certain regions, not the continent as a whole. The sample size of studies included in the analysis may have also contributed to regional and temporal bias since the regressions were weighted. The mean study size was 90 isolates for studies reporting the dhfr triple mutant genotype and 85 isolates for studies reporting the dhps double mutant genotype. However, studies examined as few as 3 isolates and as many as 453 isolates . Since studies that have small sample sizes are more likely to be skewed by rare events [Another limiting factor that must be considered when making statements regarding trends across the continent is the confounding effect of space and time. The studies reporting the frequency of the e events , and theFinally, accessibility to studies and the interpretation of data from included studies that use different reporting conventions represented noteworthy sources of bias and error, respectively. The literature search in this study was limited to the Pubmed database and Cochrane Library. Molecular studies that were not available through these sources are not reported. Studies that were included still required a level of interpretation due to differences in methodological and reporting conventions; some of which were specifically examined in this meta-analysis. However, many of these aspects simply could not be addressed within the scope this study. Since there is no established format for the collection and dissemination of molecular data, there was difficulty in determining if molecular data for the same samples was published multiple times in different articles. Efforts were made to filter out articles that reported data from isolates that were already described in a different article; however, there was uncertainty for some articles and it is possible that molecular data for some isolates were reported twice and others were excluded unnecessarily.dhfr and dhps genotypes in Africa that are known to confer anti-folate resistance. These data collectively show that the dhfr triple mutant genotype consisting of  is fixed in several parts of Africa and has been increasing, on average, over the time period from 1993 to 2006. The dhps double mutant genotype consisting of (A437G and K540E) also increased in prevalence, on average, during the same time period. However, this genotype appears to only be fixed in parts of East Africa while its prevalence in West Africa remains low. Continued drug pressure with SP may contribute to further selection of quadruple dhfr mutant genotypes and triple dhps mutant genotypes in Africa; similar to what has been observed in Asia and South America. The increasing prevalence and fixation of these mutant genotypes has historically been associated with a loss of anti-folate drug efficacy and may pose a threat to settings in Africa where anti-folates are still used to treat clinical malaria.This database, despite its limitations, provides a centralized source for information describing the frequency of mutant P. falciparum infection in women with acquired immunity and usually low density asymptomatic parasitaemia. Thus, the treatment efficacy of SP for clinical malaria in young children cannot be directly extrapolated to its usefulness for IPTp [dhps codon 581 in women receiving IPTp. In a subsequent study involving young children from Tanzania, emergence of triple mutant dhps allele with mutations in codons 437, 540 and 581 has been confirmed and this coincided with the high rate of therapeutic failure [dhps mutation at codon 581 to other parts of Africa has been presented. These findings suggest that the spread of the highly resistant triple mutant dhps allele along with triple/quadruple mutant dhfr alleles in different parts of Africa could further compromise the effectiveness of SP for the treatment of falciparum malaria and may have implications for IPTp. Therefore, systematic molecular surveillance studies coupled with assessment of drug effectiveness, including in pregnant women receiving IPTp, will be critical in the future to correlate molecular surveillance data with SP efficacy.SP is currently used in sub-Saharan Africa for IPTp. In this context, the drug is used to clear for IPTp . Howeverfor IPTp  in Muhez failure . In the A continuation of efforts to conduct systematic molecular surveillance will further provide valuable information needed to construct future iterations of resistant genotype maps and databases; however, standardized methodology and reporting conventions are critical to improve the resolution and statistical power of centralized information systems. Challenges confronted during this meta-analysis suggest that patient specific data, standardized characterization of mixed genotypes, more specific date and locality descriptions, and better design and distribution of molecular studies are all critical objectives that must be pursued in order to fulfill the vision of the centralized resistance network proposed by WWARN.The authors declare that they have no competing interestsVU and JWB conceived the original format and objectives of the meta-analysis. KMH produced an initial version of the database and genotype frequency maps. LMS and SS developed search criteria, data extraction methods, and tabular design. SS extracted and compiled the frequency data examined in the meta-analysis. SKM performed all statistical analyses and worked with SS and VU to write the first draft of the manuscript. All authors have read and approved the final draft of the manuscript.Mutant Allele Frequency (MAF) Database. The data provided show the mutant allele frequency reported in each study, organized by country in alphabetical orderClick here for fileMutant Genotype Frequency (MGF) Database. The data provided show the mutant genotype frequency reported in each study, organized by country in alphabetical orderClick here for fileArticle Database. The data provided reference each article from which frequency data are extracted, organized by study identification numberClick here for file


Gambierdiscus in the eastern Atlantic Ocean of the northern hemisphere and in the Mediterranean Sea, as well as the confirmation of CFP in the Canary Islands and possibly in Madeira, constitute other reasons to study the onset of CFP in Europe [Ciguatera fish poisoning (CFP) occurs mainly when humans ingest finfish contaminated with ciguatoxins (CTXs). The complexity and variability of such toxins have made it difficult to develop reliable methods to routinely monitor CFP with specificity and sensitivity. This review aims to describe the methodologies available for CTX detection, including those based on the toxicological, biochemical, chemical, and pharmaceutical properties of CTXs. Selecting any of these methodological approaches for routine monitoring of ciguatera may be dependent upon the applicability of the method. However, identifying a reference validation method for CTXs is a critical and urgent issue, and is dependent upon the availability of certified CTX standards and the coordinated action of laboratories. Reports of CFP cases in European hospitals have been described in several countries, and are mostly due to travel to CFP endemic areas. Additionally, the recent detection of the CTX-producing tropical genus n Europe . The que Ciguatera Fish Poisoning (CFP) is a human intoxication caused by the consumption of fish that contain ciguatoxins (CTXs) . The geoGambierdiscus. Numerous congeners of CTXs have been identified according to differences in their molecular structure. CTXs from distinct geographical origins  have been described. Some CTXs are transmitted through metabolism in the food chain. The high toxicity and diversity of CTXs, along with their presence at trace levels in fish tissue, make their reliable detection and quantification in fish flesh difficult. Numerous methodologies based upon different approaches  have been developed for CTX recognition. Their applicability not only depends on the sensitivity and specificity of the method , but also relies on effective extraction and clean-up procedures, which are necessary to prepare samples for analysis. In the present review, a selection of protocols for sample preparation prior to the application of different methodologies of CTX determination is described. A revision of the different approaches available for CTX determination, i.e., in vivo mouse bioassay (MBA), in vitro cell based assays (CBA), competitive receptor binding assay (RBA), immunological analysis, and physicochemical analysis (HPLC), are further described. The applications of these assays as routine screening tools of CTXs in fish and Gambierdiscus spp. samples are also discussed.CTXs are secondary metabolites produced by the marine benthic dinoflagellate of the genus Gambierdiscus, which are potential CTX producers, has been recorded in the Mediterranean Sea since 2003 PbTx standard for the sodium channel receptor. The use of [3H]PbTx is a necessity, as presently no tritiated CTXs is available. This is due to the infinite quantity of CTXs present in biological samples and the large quantities  of pure toxins necessary for the synthesis of tritiated standards. Most authors cited above used [3H]PbTx-3 and a few used [3H]PbTx-9 [3H]PbTx-3 (15 Ci/mmol) is prepared by the reduction of PbTx-2 with [3H] sodium borohydride according to the method reported by Poli et al. [3H]PbTx-3 is commercially available on demand and the purity of the [3H]PbTx-3 should be near 99% as determined by HPLC analysis and the stock solution should be kept at −80 °C. During the RBA, the concentration of [3H]PbTx-3 may vary between 0.5-3 nM according to authors.The RBA designed for CTXs traditionally measures the binding competition between ciguatoxin in the sample and a [H]PbTx-9 ,248,249.i et al. . Certifi3H]PbTx for receptor binding has been carried out in test tube PbTx) concentration. The concentration of synaptosomes used in the RBA varies from 40–150 μg mL−1 according to the authors PbTx-3. Competitive experiments are conducted with synaptosomes concentrations chosen in the linear portion of the curve and constant [3H]PbTx-3 with increasing concentration of cold competitor (non radiolabeled toxin or sample to test). The bound and unbound cold competitor are further separated by centrifugation or filtration, and the amount of bound [3H]PbTx-3 is measured using a liquid scintillation counter in a given volume of scintillation cocktail . Then, each competitive experiment yields a sigmoidal dose-response curve with binding parameter data such as the concentration of cold competitor that induces 50% inhibitory radiolabeled binding (IC50), Kd (dissociation constant), Ki (inhibition constant), Hill slope, and Bmax .The optimal binding conditions are checked with total binding giving no more than 10% of the total radioactivity and with non specific binding lower than 30% measured in the presence of saturating concentration of PbTx-3. Specific binding is obtained when subtracting non specific binding from the total binding. Saturation experiments are performed using constant synaptosomes concentration and increasing PbTx-3 binding. These components could interact with the synaptosome membranes and disrupt the brevetoxin binding site on the sodium channel by altering protein structure or the interaction of the protein with other membrane component +, 1,133.8 for [M + Na]+, 1,129.8 for [M + H + H2O]+ and losses of up to five water molecules . The mass spectrometric analysis obtained with IS provided similar mass spectra and fragmentation patterns to those reported previously with FAB. The technique reached a limit of detection of 1 ng of pure CTX-1, equivalent to that reported for HPLC-FLD. However, severe ion suppression effects were found when a crude lipid extract of fish was spiked with P-CTX-1 at an equivalent of 1.5 ng g−1 flesh. Nevertheless, this was the first attempt to provide a simple, sensitive and confirmatory HPLC-MS coupled technique.The use of mass spectrometry (MS) for the analysis of ciguatoxins was already considered during the first stage of characterization and structural elucidation of congener structures, mainly by using fast atom bombardment (FAB) as an ionization technique. The introduction of ionspray (IS) as ion source for MS analysis of CTXs was reported by Lewis  in 1994 . The varLycodontis javanicus) allowed determination of up to 14 P-CTXs congeners at m/z of 1,095.7 (two), 1,111.6 (six) or 1,127.7 (six), including dominant ions for P-CTX-1, -2 and -3 [4]+ and sodium [M + Na]+ adducts. It was noted that acetonitrile:water gradient modified with 1 mM ammonium acetate provided better results than modification with 0.1% TFA, as well as the use of turbo-assisted HPLC-MS, which provided a 20-fold improved signal over conventional HPLC-MS. The limit of detection allowed easy detection of 4 ng g−1 P-CTX-1 in fish flesh. Adducts of sodium, ammonium, or even potassium, as well as ions resulting of up to five water losses have also been reported for C-CTXs [HPLC-MS analysis of highly purified extracts from the viscera of ciguateric moray eels (2 and -3 . In addir C-CTXs . This chCaranx latus) that was previously purified with size-exclusion chromatography with a TSK column (Toyopearl) [4]+, [M + H]+ and [M + H − H2O]+ and [M + H − 2H2O]+ were present in significant intensities when formic acid was used as solvent modifier. On the contrary, lower intensities and absence of [M + NH4]+ pseudomolecular ion was obtained with TFA. Besides, formic acid also improved the base peak chromatogram and the MS spectrum by five-fold compared to TFA, and consequently seemed to be more adequate for CTX ionization. Reversed-phase separations were more efficient on a Zorbax C3  than on an Aquapore C18 RP 300 (1 × 50 mm), especially to separate closely eluting congeners. Chromatographic gradient was performed with acetonitrile:water (both modified with 0.1% formic acid). The analysis of toxic fractions led to determination of five new CTX congeners at m/z for [M + H]+: 1,159.6, 1,143.6, 1,157.6, 1,127.6; plus several isomers of them and of the C-CTX-1, not previously reported. The same conditions allowed determination of some of these congeners in other fish species such as grey snapper (Lutjanus griseus), grouper (Serranidae) and a black jack (Caranx lugubris) + the most abundant for the Indian congener. Further studies on red emperor led to characterization of three additional Indian ciguatoxins (I-CTX-2 to -4). I-CTX-1 and -2 showed indistinguishable masses from C-CTX-1 and ca. 60% of the toxic potency of P-CTX-1. The spectra patterns involved the typically associated ions, namely [M + H]+, [M + Na]+, [M + NH4]+, [M + H - nH2O]+, with n from 1 to 3. It was presumed that I-CTX-1 and -2 may arise from different dinoflagellates and then oxidized to I-CTX-3 and -4.I-CTXs were first reported by Hamilton et al. , isolater C-CTXs ,98,147, et al. reported coupling of HPLC with tandem mass spectrometry (MS/MS) [3 from the dominant [M+NH4]+ pseudomolecular ion and additional losses of up to three water molecules. Thus, transitions were (m/z): 1,128.7 > 1,094.0/1,076.0/1,058.0 for P-CTX-1 and 1,158.6 > 1,123.6/1,105.6 for C-CTX-1. The method provided a linear response within the working range and improved sensitivity: the limits of detection were as low as 0.04 and 0.1 ng g−1 fish flesh for P-CTX-1 and C-CTX-1, respectively. Chromatographic conditions used a reversed-phase column Vydac  eluted with linear gradient acetonitrile:water modified with 0.05% TFA.A further work from Lewis  (MS/MS) . The MS/2O]+ as precursor ion , and the response added. For P-CTX-1, two groups of transitions were monitored and the response added. The first took the molecular ion [M + H]+ as precursor ion  and the second was based upon the ammonium adduct [M+NH4]+ . Turbo ion spray conditions were identical for nebulizer gas (50 psi), turbo gas (50 psi), temperature (400 ºC), curtain gas (20 psi), and IS voltage (+5 kV). Collision energy was specifically optimized for each transition and ranged between 15–37 eV. Chromatographic conditions used reversed-phase Luna C8(2) column  and programmed linear gradient with acetonitrile:water modified with 0.1% formic acid. This procedure was successfully applied, in combination with cell-based assays, as a confirmatory technique in the study of CFP outbreaks since 1999.An 1100 HPLC (Agilent) coupled to a 4000 QTRAP hybrid mass spectrometer (Applied Biosystems) was applied for the confirmation of P-CTX-1 and C-CTX-1 on the basis of mass and retention times compared with reference materials . Workinget al. +, with subsequent loss of ammonia and successive losses of water . Soon after, Stewart et al. presented a modification of the rapid extraction method proposed by Lewis et al. with HPLC-MS/MS analysis of P-CTX-1, -2 and, -3 in ciguatera-suspect fish + for precursor ions and then the fragments at m/z 165 and 125 for product ions, according to the product ion spectra obtained intra-laboratory. The use of such alternative product ions indicates that the maximum sensitive and selective in MRM analysis should be investigated for each type of instrumentation by tuning the MS with proper CTXs standards. The advantages of using ultra-performance chromatography and separation on sub-2 μm particle size columns include enhanced efficiency, sensitivity and high-throughput analysis. In this case, retention times ranged from 3.45 to 4.00 min for the congeners analyzed, and the limit of detection that was estimated with non-certified standard solution was 10 pg loaded on column + adducts. A wide variety of congeners were determined, though P-CTX-3C, -3B, -4A, -4B and M-seco-CTX-3C added up to 97–98% of the total CTXs found in two G. polynesiensis clones. Interestingly, the toxin profiles seemed to be characteristic for each clone and their total toxin content increased with the age of culture.The production of CTXs by several strains was also investigated for different species of LC-MS/MS . After pet al. [m/z value for molecular ion [M + H]+ of 1,045 for P-CTX-3C [m/z value seems more likely to be that of the sodium adduct [M + Na]+, which can be promoted in the analysis of algal samples with relatively high salt content.During the course of this study, a potential mistake on the molecular ion associated to P-CTX-3C firstly reported by Satake et al.  has beenP-CTX-3C ,275,276.P-CTX-3C , this m/−1 and 0.1 ng g−1 for P-CTXs and C-CTXs, respectively [In summary, HPLC-MS and HPLC-MS/MS have proved to be extremely useful for the identification and characterization of new CTXs congeners around the world. Moreover, forefront instrumentation available over recent years has provided enhanced sensitivity and selectivity by measuring accurate masses or a series of fragment ions according to characteristic patterns. Such advances have opened a new era of application of mass spectrometric detection methods for unequivocal confirmation of ciguatera suspect-fish. However, taking into account the safety limits recently reported et al. [Seriola rivoliana) was captured along the Coast of the Canary Island, filleted and consumed by five family members. Symptoms appeared between 30 minutes and 28 h after consumption and resolved after several weeks, but recurred intermittently with a lower intensity for several months. The symptoms exhibited were gastrointestinal, neurological, cardiovascular and general symptoms typical of CFP such as reversal of hot and cold sensation, though the affected family members did not require hospitalization [−1. Caribbean CTX-1 was identified by LC-MS/MS in addition to two non-identified CTX-like compounds [Cases of CFP in Europe are usually associated to ciguateric seafood imported from ciguatera-endemic areas or to CFP in travelers returning from endemic CFP areas –8. In 20et al.  reportedlization . Analysiompounds . This caSiganus luridus) in 1971–1972 [Sarpa salpa) in 1988 [Siganus spp.) in 2004–2007 [Caulerpa poisoning was proposed by Chevaldonné [Epidemiological data have reported in the past various cases of ichtysarchotoxism in the Eastern Mediterranean Sea (Israel coasts) after consumption of rabbitfish . The immunoassay tested positive, but presence of CTX-like compounds has not yet been confirmed using chemical, pharmaceutical nor toxicological methods [Recent epidemiological data reported two outbreaks of ichthyosarchotoxism diagnosed as CFP, which occurred in 2007 and 2008 in Madeira Archipelago (Eastern Atlantic Ocean) 260 miles north of the Canary Islands Archipelago . In the  methods .More recently, among a total of 64 samples collected along the coast of Cameroon, three were tested toxic using the Neuro-2a CBA . This tiGoniodoma sp. [Gambierdiscus toxicus published almost two decades later [Goniodoma sp may actually constitute the first evidence of the presence of the genus Gambierdiscus in Eastern Atlantic Ocean waters. In 2004, Fraga et al. reported for the first time the presence of Gambierdiscus sp. in the Canary Islands, at a latitude 12º N of Cabo Verde [Gambierdiscus by Litaker et al. [Gambierdiscus clones isolated from the Canary Islands, in order to specify whether these Eastern Atlantic clones should be placed in a separate species group. Regarding the toxicity of the Canary Islands isolates, recent studies conducted on Gambierdiscus cells crude extracts revealed high unspecific toxicity on the Neuro-2a CBA [In 1948, a dinoflagellate reported as doma sp.  was detees later . Therefobo Verde . Based ur et al. , morpholo-2a CBA , probablo-2a CBA ,284. No Gambierdiscus spp. in the Canary Islands waters also coincided in time with both ciguatera outbreaks reported, the issue of a possible impact of Gambierdiscus blooms on public health and local fisheries in this area should also be considered. So far, there is no direct proof that these events are related, as amberjack can migrate and could become toxic far from the Canary Islands where it was captured. Moreover, other groups of benthic dinoflagellates such as Ostreopsis, Coolia, and Amphidinium commonly present in ciguateric biotopes were also detected epiphytically on benthic macroalgae in Tenerife, along with Gambierdiscus cells. Very recently, additional strains of a yet undetermined but different Gambierdiscus species have been isolated in the Canary Islands [As the first description of  Islands .Gambierdiscus populations reported in 2007 in Crete Island (Greece) [Gambierdiscus spp. could be confirmed as present since 2003 [G. toxicus type” as defined by Richlen et al. [in vitro conditions [Gambierdiscus in Crete has not coincided nor has been associated with the occurrence of CFP cases since 2003. However, it raises questions about a potential risk of CFP within this area, and to a larger extent within the Mediterranean Sea.This same observation also applies to (Greece) , which cnce 2003 . Althougn et al. , it was n et al. . Morpholn et al.  in an atnditions . They alnditions . These dOstreopsis and Gambierdiscus, in the Eastern Atlantic Ocean has posed the question of the impact of climate change on the spreading of microalgae to more temperate waters [Gambierdiscus spp. for the first time in the Mediterranean Sea [Ostreopsis spp. in the area [Gambierdiscus in the Eastern Mediterranean Sea raises the issue of its origin; lessepsian migration  or invasion via the Strait of Gibraltar are general possible hypotheses, however, further phylogenetical, taxonomical and toxicological analyses will shed light on Gambierdiscus phylogeography. Anthropogenic introduction of the species cannot be excluded, although there is no evidence or objective observation that may suggest such an origin. The fact that ciguatoxins from the Pacific are different from those from the Caribbean [Gambierdiscus and Ostreopsis share similar ecology, these genera may have similar biogeographical patterns. Distinct biogeographical patterns are observed among Ostreopsis species from the Indo-Pacific Ocean and those of the Atlantic Ocean including the Mediterranean Sea [Gambierdiscus spp. and possible risk associated to ciguatera, one may not forget that toxicity studies on several Gambierdiscus strains of different latitudinal distribution in the western Atlantic showed that in the Northern hemisphere, Gambierdiscus strains located at higher latitudes (Bahamas) were less toxic than those at lower latitudes (Caribbean) [The identification of tropical and subtropical representatives of potentially toxic benthic dinoflagellate genera, such as e waters . This hye waters , taking e waters . The detnean Sea , along wthe area ,290–293,aribbean –98 mightnean Sea ; the twonean Sea . In respribbean) .i.e., geographical expansion of toxic benthic dinoflagellates, as a possible consequence of climate change), and its impact on human health  [Gambierdiscus spp. is absent from these areas. Extrapolation of this model to the Mediterranean and Macaronesian waters suggest the importance of monitoring sea surface temperature in addition to Gambierdiscus spp. populations for the assessment of a possible onset of ciguatera in the Mediterranean Sea and Macaronesia in relation to global warming.There is increasing discussion about the link between climate variability and harmful algal blooms, (sonings) ,296–298.sonings)  particulsonings)  or organsonings) . Diversisonings) . Global sonings) . Some ofTetraodontidae, Molidae, Diodontidae and Canthigasteridae”). These regulations also consider marine toxins present in shellfish products and set the maximum permitted levels of some toxins present in shellfish products. For shellfish toxins, contrarily to ciguatoxins, European regulations clearly indicate the reference analytical methods for their detection [The presence of marine biotoxins in seafood products is regulated in order to assess the consumer’s safety. European regulations enforce specific hygiene rules for food of animal origin, including toxins, affecting human health . For CFPetection . These oetection –306 and etection .As previously emphasized, European regulations give no indication about a reference analysis method for CTXs, nor about regulatory safety limits in fishery products. This lack of specification should not be interpreted as a lack of interest, as several countries in Europe—e.g., France, The United Kingdom, The Netherlands—actually have territories in tropical and sub-tropical ciguatera endemic areas periodically affected by ciguatera. Instead, this might reflect the difficulties that laboratories dealing with ciguatera have encoutered during the last decades regarding the definition and standardization of CTX determination methods and establishment of safety limits. As for the tentative limits proposed by several authors cited herein ,119,120 The European Food Safety Authority (EFSA) is currently completing a mandate to issue a scientific opinion on ciguatoxins (Mandate M2006-0060 on Emerging toxins-ciguatoxins) in response to a question raised by the European Commission (DG-SANCO). The mandate is expected to be completed by July 2010.Of note, within the EU, national regulations specifically addressing CFP issue do exist in some countries. For example, some French overseas departments and territories are mainly located in ciguatera endemic areas . The French legislation directly incorporated the European directive, which is applicable for products imported into France from outside the EU and permits the import of certain marine species off a positive list . In FrenThe risk analysis approach is highly advisable to face CFP and establish recommendations when dealing with CFP and public safety. As for any other food intoxication, it is also very important that CFP risk analysis is coordinated by one agency or body, even if several agencies or people have to be involved.In the process of risk analysis, three major issues should be addressed: risk assessment, risk management and risk communication. Numerous reviews on CFP risk, but also addressing other CFP issues, have contributed to CFP risk assessment ,311,312.The different steps regarding risk assessment should consider hazard identification, hazard characterization, exposure assessment and risk characterization. For CFP, efforts should concentrate on CTXs identification, toxicity evaluation and epidemiology.A recent review on ciguatera fish poisoning and its’ treatment, prevention and management  resumes Risk management is often presented in four steps, which include risk evaluation, risk management option assessment, implementation of management decisions and monitoring and review of management actions. As for the risk management of CFP, different countries or regions affected have developed through the years several options and decision-making protocols that are regularly implemented . CFP manThe large number of fish species involved in CFP toxin transmission and the high degree of variability in toxin content within a given species is one of the major limitations for CFP monitoring. As a matter of fact, fish catches in CFP endemic areas are often recreational or in the hands of local fishermen working independently. This situation noticeably contrasts with shellfish products, for which most production areas are well defined in space and information provided to the producer and control of the production is easier. The availability of simple, fast and reliable tests that could be applied efficiently for the immediate screening of all individual fish on shore or in the market still needs to be achieved. Indeed, most methodologies for CFP toxin determination rely on sophisticated protocols which require long and elaborated laboratory manipulations as well as well-equipped laboratories.Gambierdiscus spp. populations within a given area and their toxin production could determine the eventual risk of CFP in the area. However, the fact that the CFP toxin trans-vector  are mobile, and the fact that toxin in fish could result from a long-term accumulation, their toxin content may not be representative of the concentration of microalgae at the time and place where the fish was caught. It is therefore advisable to invest management efforts both in preventive and palliative measures.Moreover, the density of G. toxicus and toxic fish, but also by anthropological considerations such as fishing and food habits in the population, local control and regulations. Surveys among the population can establish tawareness of CFP within a given area and bring to the surface interesting information such as the identification of the most dangerous fish for CFP within the area.As a response to the onset of CFP in Europe, it will be important to take in consideration various issues for the efficient management of CFP. Studies related to the development of toxin detection techniques and to the presence, distribution and toxicity of toxin-producing species and toxic fish are required on a long-term basis, provided that CTX standards and CTX reference materials are made available. The resulting information may identify species and fishing areas with high risk of CFP. Epidemiological data are clear indicators of the hazards of CFP within a given area. It is therefore important to favor the report of CFP cases to public health professionals and to confirm the presence of CTXs in leftovers meals, in order to facilitate differential diagnosis with other forms of seafood poisoning or fish intoxications (ichthyosarcotoxaemias). Additional effort towards the development of epidemiological studies is therefore advisable. Ethnological studies and surveys can also reveal valuable information for risk management actions and risk assessment. It is important to state that the geographical distribution of CFP is not only determined by the presence of et al. [As for the treatment of CFP, reported in Section 2.2, mannitol administration (as well as other symptomatic and supportive treatments) has been widely described; a comprehensive synthesis is presented by Friedman et al. . Adminiset al. ,62,313.etc.Risk communication strategy is a matter of serious concern: it has major impact on the different bodies and people involved, and influences the fate of hazards. A primary requirement regarding risk communication of food hazards is that the body in charge of disseminating the information regarding the risk has to be well acquainted with the exhaustive and updated scientific and technical information involved. A second issue is to define the strategy(ies) of risk communication chosen, which involves determining several factors such as the objective for such communication, the targets and the existing communication tools adapted for the situation. Risk communication has to take into consideration and predict the perception and reaction of the end-users of the message: consumers, other agencies, medical bodies, food industry, etc.) as a preventive measure to reduce cases of CFP intoxications: this is especially a very important issue in non-ciguatera endemic areas with presence of CFP caused by ciguatoxic fish imports or people travelling to ciguatera endemic areas. Selective targeted CFP risk communication can also favor transfer of information among agencies to improve CFP risk assessment, such as obtaining more accurate and reliable epidemiological data.CFP is a complex phenomenon and the risk communication strategy for CFP is far from evident . On the Gambierdiscus in the Canary Islands and in the Mediterranean Sea (Greece).Risk analysis studies are elaborate, require skilled working groups and may be costly. Considering the current state of CFP in Europe, one may question the relevance of setting up a risk analysis program in Europe. Three pieces of evidence contribute to the unequivocal settlement of CFP as a health hazard in Europe: (i) reports of CFP intoxication cases in European hospital and other medical institutions linked to fish imported from the tropics or to travelers returning from ciguatera areas; (ii) presence of fish containing CTXs in the Canary Islands and suspicion of toxic fish in Madeira archipelago; and (iii) recent reports of the ciguatera-causing dinoflagellate At this stage, it is difficult to define the current awareness of the administration in Europe regarding CFP, and one may postulate that European countries having territories in CFP endemic areas, such as France or the Netherlands, may be better acquainted. As previously outlined, European legislation takes into consideration CFP, but does not provide any elaborate information regarding the definition of toxins levels and methods for toxin determination.Having this evidence in hand, and being aware that it is also important not to magnify the risk perception associated with the consumption of fishery products, risk analysis for CFP in Europe is a necessity. This is particularly apparent when we take into consideration that travelling to tropical areas is not anecdotic and that the distribution of ciguateric fish and CTX-producing microalgae might be expanding in European waters. Risk analysis for ciguatera in Europe should be addressed by implementing efforts proportional to the present situation, maintaining the commitment to assure an extensive assessment of the current CFP situation in Europe in order to correctly define the management and communication strategies that should be developed.It may be expected that a risk analysis study on CFP to define detailed risk assessment and management plans as well as establish a proportionate risk communication strategy, may be considered in the future. Some preliminary and basic recommendations at this stage on possible priority actions needed in Europe are presented: Preliminary needs:- Favor that the competent authority will lead actions related to risk analysis for CFP, in coordination with the different agencies implicated.- Identify research agencies implicated in CFP to improve risk analysis.- Provide the correct framework within the EU to have competent laboratories for the analysis of CTXs in food.Risk Assessment:- Systematically record possible cases of CFP with strict identification of symptoms and nature and origin of suspicious food.Seriola spp. in the NE-Atlantic and consider extending analysis to other fish located in areas with presence of Gambierdiscus spp.- Structural and toxicological characterization of CTXs and other toxins present in food and microalgae associated with CFP. Pursue recognition of CTX presence in Gambierdiscus spp.- Follow benthic microalgal distribution of hazardous species with special focus on - Evaluate exposure to CTXs.Risk Management:- Revise current legislation on CFP and foresee the set-up of expert analysis groups to identify deficiencies and future needs.- Establish preliminary monitoring programs in areas where CFP is present.- Centralize records of CFP cases in Europe for epidemiology surveillance.- Establish CFP treatment protocol(s).- Work in association with food import companies, local fisheries agencies in ciguatera areas and tourism agencies to establish action and communication protocols.Risk communication- Define a protocol for a widespread communication strategy to the general public to be used in case of a ciguatera episode.- Define and implement selective communication targeted at agencies and specific bodies.i.e., toxin profiles, implicated fish species, local food habits, etc. However, there is also a lack of consensus world-wide on several CFP issues, especially regarding the methods for CTX determination in food. Several factors may be responsible for the present situation: (i) lack of certified standards and reference material, (ii) lack of a coordinated action among laboratories, and (iii) frame of applicability of the method (rapid screening versus accurate and confirmatory methods). A worldwide coordinated action could certainly improve adopting harmonized strategies to face-up to CFP and especially favor the establishment of a validated reference method for CTX determination in food. Such a method could be be used for unequivocal identification and accurate quantification of CTXs and as reference for the validation of more rapid screening methods. To achieve this objective, obtaining certified standards and reference material, exchanging samples and setting-up inter-laboratories comparative studies is needed. Possibly, LC-MS/MS is the most probable candidate to become the reference method for CTXs once certified standards are available, a standard operation procedure is validated, and the required limits of detection are guaranteed. Additionally, functional assays such as CBA or RBA have arisen as promising techniques for high-throughput screening of samples and therefore efforts should be also carried out to validate them.CFP is managed according to national or regional strategies, often dependent on the regional characteristics of CFP, This coordinated action could also consider a common risk analysis strategy to improve risk assessment, risk management and risk communication, in order to reduce the impact of CFP worldwide. If one may expect that this rationale would be shared by the overall scientific community and managers dealing with CFP, one may conclude that the present key issue would be to identify the platform or forum that could lead this coordinated action. Certainly all co-authors herein would favor international cooperation in this matter.CFP, mainly distributed in tropical and subtropical areas, is already affecting regions in temperate latitudes such as Europe. As CFP intoxications are being reported in European hospitals, methods for CTX identification are needed to confirm these cases. Once this (these) method(s) are available, statistics and epidemiological records regarding CFP affected people in Europe should also improve.Field studies should also contribute toward better understanding the impact of CFP in Europe. Understanding the distribution of CFP toxin-producing benthic dinoflagellates and evaluating CFP toxins in fish and microalgae should allow a better prediction of CFP risk. Additionally, this information would contribute to hypotheses regarding the possible expansion of CFP towards northern latitudes as a consequence of climate change. Improving detection of CTXs and understanding CFP spatial distribution will contribute to improve the risk analysis of CFP in the European geographical area.


Image-based B-mode ultrasound has gained popularity in major studies as a non-invasive method of measuring cardio- and cerebrovascular risk factors. However, none of the major studies appears to have paid sufficient attention to the variation in end diastolic wall process. By using sequentially based analyses (SBA) of Intima-Media Thickness (IMT), the general purpose of this study was to show that the current image based (ECG tracked) analysis (IBA) has some major variations and might underestimate the true relations for cardiovascular events and stroke for IMT measurement.The study group consisted of 2500 healthy male subjects aged between 35 to 55 years. 4 sequences (300 images) were analyzed per subject. 750,000 images were analysed throughout the course of this study.lower mean, maximal, and minimal values for IMT in CCA than for SBA. The correlation analysis between IBA and SBA with the cardio- and cerebrovascular risk factors showed a higher correlation of SBA for all risk factors. The Pearson coefficient was 0.81, p < 0.01, for SBA versus Framingham CHD risk level (FCRL) and 0.49, p = 0.01, for IBA versus FCRL.IBA showed significantly IBA did not measure the true maximal values of the IMT in this study. Together with the correlation analysis, this indicates that IBA might underestimate the true relations for IMT and risk factors. Over the last few years, numerous studies have linked carotid Intima Media Thickness (IMT) and IMT progression with prevalent symptomatic coronary artery disease (CAD) -5 and ceO'Leary and colleagues  reportedIt seems logical to develop additional ultrasound methods to measure the risk of cardio- and cerebrovascular events and stroke more precisely.The carotid artery seems to be a useful window for cardio- and cerebrovascular risk, with ultrasound able to provide unique information on morphological, hemodynamic and elastic properties. The majVarious ultrasound techniques have been used to detect and track vessel wall motion – manual as well as computer based measurements. Computational techniques have mainly been based on analysis of the B-mode greyscale images , M-mode All methods – manual and automatic – have one main part in common; all methods use an end diastolic ECG tracked image . Is thisIn heart rate regulation, baroreflex plays a very important role, and is commonly related to carotid baroreceptor function. Systemic changes of the blood vessel wall and heart contraction may affect carotid artery pulse wave phase characteristics: carotid artery dilates more or less, decreases or increases the impulsation from baroreceptors. Because of that, heart rate variability changes in the baroreflex band of the frequency spectrum. Murgo et al ,23 dividMost of the major studies about IMT in major journals,24 have The aim of this study was to evaluate the correlation of image based IMT measurement to sequentially based analysis and cardio- and cerebrovascular risk factors.The study group consists of 2500 healthy male subjects (HS) aged between 35 to 55 years (age: 46.1 ± 5.6 years). All subjects underwent a detailed interview, completed a questionnaire (26) to evaluate their physical activity and food habits, a clinical examination, an ultrasound examination of the carotid tree, and a blood test for the lipid profile.All subjects were free of cardiovascular disease as determined by medical history. The characteristics of the study population are given in Table Subjects were advised to avoid caffeine intake for 8 h before the study and to maintain their usual lifestyle  between studies. None of the subjects took any medications.The research was carried out in accordance with the Declaration of Helsinki (1989) of the World Medical Association, and has been approved by the Freiburg University Research and Ethics Committee.After at least 15 minutes rest in supine position, each patient was scanned by six experienced ultrasonographic observers.We used a Toshiba SSA-380A, Japan, ultrasound scanner with high resolution 10 MHz transducer and an aperture of 32 mm. The carotid tree was scanned the transducer positioned longitudinal to the CCA. The lumen was maximised and the position changed until the typical double lines of the intima and adventitia boundaries of the arterial wall could be seen. Both, near and far, boundaries could be seen in the scan.We stored one sequence for each subject for the lateral and for antero-lateral view for left and right CCA (4 sequences per subject) with the simultaneously recorded ECG at the top of the R-wave on digital disk  Figure .Each sequence contains a time period of 3 seconds including at least 2 heart cycles. Each sequence was stored with 25 Hertz, which included 75 images per sequence. Additionally, all examinations were stored on videotape in order assist in the interpretation, if necessary.After all we used 300 images per subject for the analysis . In the course of this study, 750,000 (2 × 2 × 75 × 2500 subjects) images for the sequence and 10,000 (2 × 2 × 2500 subjects) single images were analysed.All measurements and analyses were done by one experienced reader blinded to all clinical information. The reader visually controlled stored sequences of each subject.For image based analysis (IBA), the reader chose the image from the digital videotape of the patient during the ultrasound examination with the R-wave in the ECG signal. This is the standardized procedure actually recommended by the recent consensus papers for IMT measurement.This image was analysed for Image based Analyses of IMT measurement mIMT is defined as the mean value of the total IM layer in a chosen segment.Maximal Intima-Media Thickness (maxIMT)maxIMT is defined as the maximal value of the total IM layer in a chosen segment.Minimal Intima-Media Thickness (minIMT)minIMT is defined as the maximal value of the minimal total IM layer in a chosen segment.Maximal Mean Intima-Media Thickness (MmIMT)MmIMT is defined as the maximal mean value of the total IM layer in a chosen sequence and segment.Maximal Maximal Intima-Media Thickness (MmaxIMT)maxIMT is defined as the maximal maximal value of the total IM layer in a chosen sequence and segment.Minimal Intima-Media Thickness (MminIMT)MminIMT is defined as the maximal minimal value of the total IM layer in a chosen sequence and segment.For all subjects, the individual CHD risk level was estimated based on the Framingham CHD score sheets presented by Wilson et al. . This scAll data were calculated using the software program SPSS  for Windows, Version 12. The arithmetic mean and the standard deviations (SD) were used for descriptive statistics.Pearson's correlation coefficient was used to measure the strength of the linear relationship between IMT and WI A Bland & Altman analysis was performed to describe reproducibility of the method. The agreement for the reproducibility of WI was accepted if the upper and lower boundaries of the 95% confidence interval for WI covered zero.A total of 2500 subjects were examined in the study, including a total number of 750,000 images. Anthropometric data, resting heart rate, and blood pressure for both groups are shown in Table significantly lower mean , maximal  and minimal  values for the left, right, and combined CCA than for sequential based analysis .1) Sequential analysis could detect higher values in vivo situation of the maximal IM layers than image based IMT measurement (see 2) Sequential analysis was significantly different from image based analysis3) The results of correlation analyses may lead to the assumption that current IMT studies on image based analysis might underestimate the true relations of individual risk for atherosclerosis .First it is important to emphasize that this study used the same computer based detecting algorithm for the image based analysis as for the sequential based analysis. Using the same algorithm (25) reduces the errors of detecting the correct layers to a minimum.The image used for the image analysis (IBA) was part of the sequence used for the sequential analysis. This is important to emphasize. Using this procedure – we used the same detection algorithm, the same reader, and the same video sequence for the analysis of the image based and sequence based analysis – we reduced the variation and reading errors for the IBA and the SBA analysis to a possible minimum. One variation left is the possibility that the differences seen in IMT measures between one base image and a sequence of images might be due to motion during the acquisition of the carotid artery images. The ultrasound observer tried very hard to get a stable scan sequence during the 3 seconds of data recording. Due to our ultrasound procedure we think that this possibility is very small.The remaining difference can be assumed as true difference of both methods.In this study we found a significant difference between IBA and SBA. The results of Bland-Altman analysis showed that both methods did not detect significantly the same values of IM layers. The image based analysis (IBA) measures a significantly lower IMT value than the sequence based analysis. What is the reason for the difference?A detection error can be excluded, since we used an automatic system. The obvious and logical answer is that image based analysis did not measure the maximal value of an Intima Media layer during a heart cycle .However, in epidemiology, it is not the absolute value that is important, but how it ranks subjects as having high or low risk. For example, the CLAS study had an IMT value of 0.60 mm whereas this was a very high risk group. The ARIC study had mean levels of 0.70 mm in middle aged subjects with lower cardiovascular risk. However, the ability to rank subjects using the method is also very relevant. The differences between the results of both studies might be explained by the different detection methods and by the end diastolic time variations.The CLAS and ARIC studies showed the possibility of IMT measurement as a parameter for to evaluate cardio- and cerebrovascular risk, but they also showed the lack of the actual IMT measurement protocols.Is a person with a mean IMT of 0.6 mm a person with a higher risk for cardio- or cerebrovascular disease or not? To our knowledge there is no data available which can answer this question. All major studies differ significantly with regard to the baseline values of their subjects. The main reason for this difference might be the measurement method. The answer cannot be solved by the current protocols for image based analysis due to the large number of variations and end diastolic variation errors.Using a large number of subjects, as was recently done in major IMT studies, can decrease the variation and end diastolic variation errors of other influences on the measurement. Due to the large number of subjects, the statements of the major studies are correct that IMT is a proven parameter of atherosclerotic processes in arteries.We added a correlation analysis to our study to evaluate the correlation of IBA and SBA to the Framingham CHD risk level and other proven risk parameters. We found a weak to moderate correlation for IBA and a strong association for the SBA for the risk factors. Figure However, in this study, we hoped to show that IBA might underestimate the true relations. This statement has also been shown in histopathological studies -31, but In conclusion, this study shows that sequential analysis has a higher correlation to risk factors than image based analysis. We showed that the image based analysis might underestimate the true relations of cardio- and cerebrovascular risk.We suggest using sequential analysis as a method for a robust measurement of IMT and cardiovascular risk.MS carried out the ultrasound examinations and drafted the manuscript, JH drafted the manuscript, JS developed the computer algorithm, DS carried out ultrasound examinations, AN read the manuscript, HB developed the algorithm and AS-T helped with the study design. All authors read and approved the final manuscript.


The risk of sporadic colorectal cancer (CRC) is mainly associated with lifestyle factors, particularly dietary factors. Diets high in red meat and fat and low in fruit and vegetables are associated with an increased risk of CRC. The dietary effects may be modulated by genetic polymorphisms in biotransformation genes. In this study we aimed to evaluate the role of dietary factors in combination with genetic factors in the different stages of colorectal carcinogenesis in a Norwegian population.GSTM1, GSTT1, GSTP1 Ile105Val, EPHX1 Tyr113His and EPHX1 His139Arg), and risk of colorectal carcinomas and adenomas. Odds ratio (OR) and 95% confidence interval (95% CI) were estimated by binary logistic regression.We used a case-control study design  to test the association between dietary factors  genetic polymorphisms in biotransformation genes  of the EPHX1 codon 113 polymorphism. An association was also observed for the EPHX1 codon 113 polymorphism in the low-risk adenomas, although not as obvious.A higher ratio of total meat to total fruit, berry and vegetable intake was positively associated with both high and low-risk adenomas, with approximately twice the higher risk in the 2Although, the majority of the comparison groups are not significant, our results suggest an increased risk of colorectal adenomas in individuals for some of the higher ratios of total meat to total fruit, berry and vegetable intake. In addition the study supports the notion that the biotransformation enzymes GSTM1, GSTP1 and EPHX1 may modify the effect of dietary factors on the risk of developing colorectal carcinoma and adenoma. Colorectal cancer (CRC) is one of the most prevalent diseases in the industrialized Western countries. In Norway, with one of the highest incidence rates in the world, approximately 3500 new cases of CRC are diagnosed every year .Epidemiological studies have attributed more than 85% of CRCs to environmental factors ,3, partiEPHX1) (phase I) and gluthatione S-transferases (GSTs) (phase II), all with known polymorphisms [It is widely known that humans differ in their susceptibility to cancer. Difference in carcinogen metabolism might explain the differences in cancer susceptibility especially as most cancers are influenced by environmental factors . Dietaryorphisms ,17.EPHX1 gene has a dual function and catalyzes the hydrolysis of arene, alkene and aliphatic epoxides to less reactive and more water-soluble dihydrodiols, but activates some polycyclic aromatic hydrocarbons into a more carcinogenic form [113His) of the EPHX1 gene decreases in vitro enzyme activity by 40%, whereas a histidine to arginine substitution in exon 4 (His139Arg) increases in vitro enzyme activity by 25% [The nic form . A tyrosy by 25% .GSTM1 and GSTT1 deletion polymorphisms ("null" genotypes) resulting in absence of the GST μ1 and GST μl enzymes [GSTP1 Ile105Val polymorphism resulting in a less active enzyme [GSTs are responsible for the glutathione conjugation and detoxify a number of various reactive species . Genetic enzymes , and thee enzyme .Several lines of evidence support the adenoma – carcinoma sequence as the major path for colorectal carcinogenesis. The present study aimed to investigate the role of dietary factors (meat and fruit/vegetables) in combination with genetic factors in the different stages of colorectal carcinogenesis in a Norwegian population.The KAM cohort  is based on the screening group of the Norwegian Colorectal Cancer Prevention study (The NORCCAP study) in the county of Telemark , and a sThe KAM biobank consists of blood and tissue samples from 234 CRC cases , 1044 individuals identified with polyps in the large intestine  and 400 controls, defined as individuals with normal findings at flexible sigmoidoscopy screening. Adenoma cases and controls were all drawn from the NORCCAP study. All of the participants completed a questionnaire on demographics, health status, dietary and smoking habits, alcohol consumption, physical exercise and occupation. The questionnaire contained information on a family history of adenomas and carcinomas, and the cases and controls included had no known personal history of genetic predisposition.Diet was assessed in cases and controls using an extensive diet and lifestyle questionnaire, modeled on a questionnaire developed and validated for an American breast cancer study -28. It wThe adenocarcinomas were collected prior to chemo- or radiotherapy treatment. Two specialists in histopathology examined the histology of the adenomas independently in order to determine the tumor stage as mild, moderate or severe dysplasia. They reached the same conclusion in all cases.Diminished numbers of available cases in the KAM biobank and analyzed cases are due to loss of samples during preparation and/or lack of information from the questionnaires. The 53 hyperplastic polyps were not included in the present analysis. A high-risk adenoma was defined as an adenoma measuring = 10 mm in diameter and/or with villous components and/or showing severe dysplasia .Genomic DNA was isolated from blood samples according to standard procedures  with minGSTM1 (null), GSTT1 (null) and GSTP1  genes were analyzed simultaneously by multiplex PCR as described by Nedelcheva Kristensen et al.[Polymorphisms of the en et al..EPHX1 His139Arg polymorphism (rs2234922) was analyzed by a PCR-RFLP (restriction fragment length polymorphism) method as described by Hasset et al.[The et et al..EPHX1 Tyr113His polymorphism (rs1051740) was carried out using the TaqMan allelic discrimination assay on a Sequence Detection System ABI 7000 (Applied Biosystems), using the primers forward: 5'-TCT CCT ACT GGC GGA ATG AAT T-3', reverse: 5'-CTG GCT GGC GTT TTG CA-3' and the probes T-allele: 5'-VIC-TCT CAA CAG ATA CCC TCA-MGB-3', C-allele: 5'-6-FAM-TCA ACA GAC ACC CTC A-MGB-3'. The polymorphism was determined in a 12 μl reaction containing 1× MasterMix, 200 nM of each probe, 900 nM primers, and ca 50 ng of genomic DNA. Cycling conditions were as follows: 50°C for 2 min, 95°C for 10 min, and 45 cycles of 95°C for 15 s and 60°C for 1 min. Controls were included in each run, and 10% of the samples were retyped with identical results.Genotype analysis of the Eight summary variables were created from the individual dietary items in the food frequency questionnaire to describe the intake of red meat, processed meat, total meat, raw vegetables, boiled vegetables, fruit and berries, total fruit, berries and vegetables and the ratio between total meat and fruit, berry and vegetable intake. For each individual dietary factor, the frequency and portion size were multiplied to obtain the amount in grams per day. The summary variables were then divided into quartiles based on distribution of consumption among controls.2 test for categorical variables and the Mann-Whitney test for continuous variables. P-values < 0.05 were considered significant. Odds ratios (ORs) and 95% confidence intervals (CI) were calculated using binary logistic regression analysis to assess the relationship between the dietary factors, each polymorphism and colorectal carcinoma or adenoma cases. Crude ORs were calculated, adjusted for age and gender only. In addition multiple logistic regression analyses were performed with an adjustment for age, gender, smoking dose, alcohol consumption, and when appropriate total meat consumption and total fruit, berry and vegetable consumption. In addition the modifying effect by genotype on dietary exposure  was studied by stratification in a two by four table. All analyses were carried out using the statistical analysis software SPSS  12.0.1 for Windows.Differences in characteristics between the control group and all the case groups were assessed using the χSelected characteristics of cases and controls are shown in Table The distribution of consumption of the dietary factors in quartiles is presented in Table rd quartile, OR of 1.57 (95% CI 1.00–2.46). No significant results were observed for total fruit, berry and vegetable consumption. The ratio of total meat to total fruit, berry and vegetable intake was positively associated with both high and low-risk adenomas in the 2nd quartile (and 3rd quartile for low-risk adenomas) with approximately twice the higher risk compared to the lowest quartile (ORs of 2.59 (95% CI 1.31–5.14) and 1.73 (95% CI 1.08–2.75), respectively). We have also tested the dietary exposures as binary variables, but these results gave less information than the chosen model (data not shown).No significant trends were observed in the data of Table GSTM1 null allele, GSTT1 null allele, GSTP1 105Val allele, EPHX1 113His allele and EPHX1 139Arg allele among the controls were respectively, 0.52, 0.13, 0.37, 0.32 and 0.21 , Ptrend = 0.01. No significant associations were seen between the EPHX1 139Arg allele and carcinomas and adenomas. Since the strongest association seems to be with the Arg/Arg genotype we have compared this genotype with the combined His/His and His/Arg genotypes as a reference. The Arg/Arg genotype was associated with decreased risk of low-risk adenomas only, OR of 0.48 (95% CI 0.23–0.98) (data not shown). We have also tested the combined effect of the two polymorphisms in EPHX1 . Predicted mEPHX activities were classified as normal, fast and slow [The frequencies for the and slow . The samTable 4) was observed in individuals with the GSTP1 codon 105 variant allele . In addition a positive association between ratio (Q2) and CRC risk was more obvious in individuals with the EPHX1 codon 139 common allele .No significant trends were observed. For carcinomas we did not find any significant association with ratio of total meat to total fruit, berry and vegetable intake, and the combination with genotype gives few significant results. A reduced risk  was also associated with an increased risk of high-risk and low-risk adenomas in individuals with the GSTM1 common allele and the GSTP1 codon 105 variant allele .A higher ratio of total meat to total fruit, berry and vegetable intake was positively associated with colorectal adenomas. For the high-risk adenomas this positive association was more obvious for the common allele  of the GSTT1 polymorphism on the ratio of total meat to total fruit, berries and vegetables intake was not possible to perform due to low sample size.Statistical analysis of the modifying effect by the Sporadic CRC is a consequence of multiple risk factors, among which the interaction between environmental and genetic factors is of particular interest. In this case-control study we investigated whether the consumption of meat and fruit, berries and vegetables or selected genotypes was associated with risk of developing colorectal carcinomas and adenomas alone or if the genotypes could modify the results.We found only a weak positive association between total meat consumption and low-risk adenomas. Lack of significant association with the cancer group and the high-risk adenoma group, may be due to small sample size. There appears to be consistent evidence from epidemiological data that intake of red meat is positively related to risk of developing CRC ,9. EnhanWe did not detect any association between fruit, berry and vegetable intake and risk of developing colorectal carcinomas or adenomas. The association between fruit and vegetables consumption has not been consistently proven in other studies. The majority of case-control studies show an inverse association between intake of vegetables and colon cancer risk, while prospective studies show no association . Distribnd quartile  compared to the lower quartile. This result may apply to the general Norwegian population which has little variation in intake of meat, fruit, berries and vegetables. The result is in agreement with the result of Turner et al.[The ratio between total meat and total fruit, berry and vegetable intake yielded an increased risk of high and low-risk adenomas for the 2er et al. who shower et al. who has GSTM1, GSTT1 and GSTP1 polymorphisms were not associated with risk of colorectal carcinomas and adenomas in this study. This is consistent with the results in some studies [The  studies ,43, but  studies .EPHX1 113His allele was associated with a decreased risk of low-risk adenomas. The EPHX1 codon 139 polymorphism was not associated with risk of carcinomas and adenomas in this study. Contradictory data have been provided by different studies investigating the EPHX1 codon 113 and EPHX1 codon 139 polymorphisms in relation to the risk of colorectal carcinomas and adenomas [The adenomas -50.GST and EPHX1 polymorphisms in CRC susceptibility. Since the enzymes have detoxifying activity, it would be expected that, rather than affecting the risk of cancer per se, they would modify risk when exposed to potential carcinogens [There is conflicting evidence regarding the role of cinogens .GST and EPHX1 modify colorectal carcinoma and adenoma risk in relation to ratio of total meat to total fruit, berry and vegetable intake, we stratified the case groups based on the most common allele or any polymorphic allele.To evaluate if the genetic polymorphisms in EPHX1 codon 113 in adenomas and EPHX1 codon 139 in carcinomas. In our study, for both the EPHX1 codon 113 and EPHX1 codon 139 polymorphisms, the increased risk associated with a higher ratio of total meat to total fruit, berries and vegetables was confined to individuals homozygous for the most common alleles . To our knowledge no studies have been published on the association between GST and EPHX1 polymorphisms and the ratio of total meat to total fruit, berry and vegetable intake, but some studies have looked at the gene-diet interaction with meat and fruit/vegetables separately. There are few reports on the association between EPHX1 polymorphisms and dietary factors. Cortessis et al.[EPHX1 activity  variant at exon 3 and the R (arginine) variant at exon 4), whereas Ulrich et al.[EPHX1 and CRC and adenomas did not find evidence for an interaction with meat intake [EPHX1 113Tyr allele  [The most convincing evidence of a modifying effect by genotype on dietary exposure was found between is et al. reportedch et al. reportedt intake ,47,48,50 allele) , but due allele) . While E allele) ,53. EPHXGSTP1 and GSTM1 genotypes on the ratio of total meat to total fruit, berry and vegetable intake in adenomas. The increased risk associated with a higher ratio of total meat to total fruit, berries and vegetables is restricted to individuals with the GSTP1 codon 105 variant allele , and to the GSTM1 common allele. Since there are no significant trends and only a few significant single results caution is required in interpretation of our findings.In addition we found weak evidence of a modifying effect by the GSTM1/T1 null genotypes and the GSTP1 105Val allele [GST variant could directly reflect the slower processing of genotoxic compounds associated with meat intake. In addition a high ratio of meat to fruit, berries and vegetables may increase the risk of carcinogenesis.Studies have shown that higher levels of DNA damage are associated with the l allele ,55. The Isothiocyanates (component of fruit and vegetables) are substrates for GSTs -58 and tAs with any case-control study, there is a possibility of bias in recall of diet leading to spurious associations. But for the assessment of gene-environment interactions this is not an issue, since there is no corresponding risk of bias in relation to genotype. We cannot exclude the possibility that the associations may be confounded or modified by other genetic or dietary factors. The CRC cases and controls have not been matched by age, which may affect the results in our study. However, the controls were recruited from the same population as the adenoma and carcinoma cases. Further, our controls have been screened and found polyp free by flexible sigmoidoscopy and the risk of any of them having colon cancer at the time of inclusion is not very likely. It has been estimated that less than 3% of individuals with no adenoma at flexible sigmoidoscopy are likely to harbor an undiagnosed proximal advanced colorectal neoplasia .Due to the high number of multiple tests and comparisons in the present study, some statistically significant associations have undoubtedly occurred by chance, and caution is required in their interpretation. The results thus need to be verified by further studies with particular focus on the ratio between meat and fruit, berry and vegetable intake and the possible modifying effects of biotransformation genes.Although, the majority of the comparison groups are not significant, our results suggest an increased risk of colorectal adenomas in individuals for some of the higher ratio of total meat to total fruit, berry and vegetable intake. An unbalanced consumption of meat and fruit, berries and vegetables may lead to accumulation of DNA damage and thus increased risk of cancer development. In addition the study supports the notion that the biotransformation enzymes GSTM1, GSTP1 and EPHX1 may modify the effect of dietary factors on the risk of developing colorectal carcinoma and adenoma.The author(s) declare that they have no competing interests.CFS prepared the first draft of the paper. She prepared, with contribution from MS, the dietary data for statistical analysis. She did the data analysis and participated in genotyping of the samples. MS also contributed to the manuscript and with advice in the data analysis, and she participated in genotyping of the samples. AH advised on handling of the dietary data and statistical analysis. GH together with TG and KMT were responsible for designing and administering the NORCCAP clinical trial. In addition GH and TG provided advice on the statistical analysis. ILH participated in collection and quality control of the questionnaires. EHK initiated the KAM study and also organized it. She contributed with advice on data analysis and was responsible for the revisions of the manuscript. All authors discussed the results, contributed to interpretation of the results and the final manuscript.The pre-publication history for this paper can be accessed here: