AB0 blood group is an inherited characteristic that has been associated with the incidence as well as the prognosis of several malignancies. The aim of the current study was to clarify the role of the blood group in cancer epidemiology and clinical outcome of patients with prostate cancer (PCa).Data from 3,574 patients undergoing radical prostatectomy between 2009 and 2010 at a single European institution were retrospectively analyzed. The correlation of AB0 and Rhesus blood group with PCa-related characteristics and oncological outcome were evaluated using univariable and multivariable Cox proportional hazard models.p > 0.05).Median follow-up was 36.9 months. The overall distributions of AB0, as well as Rhesus blood groups among patients with PCa, did not differ from the distribution observed in the normal population. There was no significant association between AB0/Rhesus blood groups and Gleason score, prostate volume, surgical margin, pT-stage, pN-status, or preoperative prostate-specific antigen level. In multivariable Cox regression analysis, no statistically significant correlation between AB0/Rhesus group and biochemical recurrence was observed (all Our data suggest no relevant association of AB0/Rhesus blood group with adverse clinicopathological tumor characteristics or oncological outcome after surgery in contrast to several other malignancies. Prostate cancer (PCa) is the most common malignancy in Western countries and the second leading cause of cancer-related deaths in males . The mosAB0 blood group antigens (ABH) are glycoproteins expressed on the surface of red blood cells. The phenotypic A and B antigens are terminal carbohydrates synthesized by the addition of single sugars catalyzed by a series of specific glycosyltransferases. Phenotype 0 is characterized by the absence of A and B glycosyltransferases, so that only a protein backbone, the H antigen, is present. Red blood cell antigens have various functions, including membrane structural integrity, transportation of molecules through membranes, and adhesion . Along wBased on the current evidence of the predictive role of the blood group in several malignancies, we hypothesized that the type of blood group may be used as an additional parameter, which facilitates the assessment of risks and helps to estimate prognosis after surgery in patients with PCa. In current literature, there is only limited clinical evidence available regarding blood group and PCa in general, which demonstrated controversial results –14 MoreoTherefore, the aim of the present study was to add further information to the ongoing debate by analyzing the correlation of AB0/Rh blood group and its distribution within patients with PCa, tumor pathology, recurrence, and mortality in patients with localized PCa undergoing RP by using a large single center dataset of 3,574 patients.n = 8), leaving 3,574 patients available for analysis.The study was approved by the institutional review board. A total of 3,582 patients with clinically localized PCa who underwent RP at our institution between 2009 and 2010 were screened for study inclusion. Those with incomplete data sets and missing follow-up were excluded  or robotic-assisted laparoscopic approach (n = 354), as described previously , prostate volume , Gleason score (specimen), pT-stage, pN-status, surgical margin, BCR, and mortality after RP. Patient follow-up consisted of PSA testing every 3–6 months during the first 2 years after RP, BCR was defined as PSA level ≥0.2 ng/ml.t-tests. In Cox regression multivariable analysis (log-rank tests), the impact of blood group, preoperative PSA, Gleason score, pT-stage, pN-status, and surgical margin on BCR-free survival were assessed in order to evaluate the prognostic values for survival. The probability of BCR-free survival was compared in 0, A, B, and AB groups using Kaplan–Meier analysis and the log-rank test.The relationship between AB0 blood group and clinicopathological variables were assessed using chi-squared tests and p-values <0.05 were considered statistically significant. Statistical analyses were performed with JMP software v9.0.2  and R v2.13.1 .All tests were two-tailed and A total of 3,574 patients with clinically localized PCa were eligible for final analyses, the median age at surgery was 65 years (range: 38–80) and the median follow-up was 36.9 months Table .The distribution of AB0 blood group among patients with PCa prior to RP was as follows: 0 in 1,350 (38%), A in 1,605 (45%), B in 445 (12%), and AB in 174 (5%) patients, 83% of the patients were Rh positive Table : 0 in 41p = 0.033). Patient population with AB also showed a moderately increased ratio of negative Rh factor (21.8%) in comparison to the other blood groups   and 63.3% of patients had preoperative PSA levels within the range of 4–10 ng/ml. pT2 tumors were detected in 68.7% and negative margins were achieved in 81.3% of patients. The median prostate volume was 40 ml and lymph node involvement was diagnosed in 224 (6.3%) patients (Table p < 0.05) (Table p = 0.74).According to multivariable cox regression analysis, preoperative PSA, Gleason score, pT-stage, pN status, and surgical margin significantly and independently correlated with BCR-free survival  Table . However5) Table . We furt5) Table . Even if5) Table A,B. MorePossible associations between AB0 blood group and the risk of some epithelial malignancies, including pancreatic-, colorectal-, and gastric cancer have been reported frequently , 20. MorSeveral decades ago, immunohistochemical studies indicated that patient-derived PCa cells show different patterns of ABH expression than normal/benign prostate cells . Since bA recent study published by Markt and colleagues  analyzedn = 279) (p = 0.026). In this study cohort, 46.3% of patients had positive margin status despite of a clinical stage of T1c in 74.2% of the patients. The pathological stage (pT) was not mentioned.These results are in line with a study run by Kvist and colleagues, which demonstrated no correlation between AB0 blood type and the survival of patients with PCa managed with watchful waiting. This study was mainly limited by the small case number (n = 279) . Interesn = 279) . In the Our retrospective cohort of 3,574 patients with clinically localized PCa is the largest cohort being analyzed so far to determine a possible impact of both AB0 and Rh blood groups on tumor characteristics and outcome after RP. In the present study, the majority of patients had pT2 tumors (68.7%) and 18.6% had positive margin status. Using these data, we could not show any significant impact of AB0/Rh blood groups on clinicopathologic characteristics  and BCR-free survival after curative RP. Moreover, when performing a subanalysis with patients with positive or negative margin status only, the influence of AB0/Rh on BCR remained insignificant. Hence, we could not reproduce the results of the Japanese study . In contp = 0.01) could be possibly boosted due to the rarity of Rh-negative AB type, which prevents a statically significant detection of a correlation within this subgroup. Finally, information on genotype of the respective blood group was missing.There are several important limitations to our study. First and foremost are the limitations inherent to retrospective analyses and the lack of a control group. Furthermore, our study cohort predominantly contains localized PCa with low- to intermediate risk profile  allowing pre-selection of patients with favorable prognosis. In addition, the overall distribution of AB is—in contrast to the other blood groups—generally low abundant (4.9%). Some observation such as the moderately increased ratio of negative Rh factor (21.8%) in patients with AB in comparison to the other blood groups .


Staphylococcus aureus encoding the fluorescent reporters mAmetrine, CFP, sGFP, YFP, mCherry and mKate. The expression is driven either from the sarA-P1 promoter or from any other promoter of choice. The reporter can be inserted markerless in the chromosome of a wide range of S. aureus strains. The integration site chosen does not disrupt any open reading frame, provides good expression, and has no detectable effect on the strains physiology. As an intermediate construct, we present a set of replicating plasmids containing the same fluorescent reporters. Also in these reporter plasmids the sarA-P1 promoter can be replaced by any other promoter of interest for expression studies. Cassettes from the replication plasmids can be readily swapped with the integration vector. With these constructs it becomes possible to monitor reporters of separate fluorescent wavelengths simultaneously.We present integration vectors for  Staphylococcus aureus is one of the most problematic pathogens for human health, able to cause skin and soft tissue infections, as well as serious invasive diseases such as sepsis, endocarditis, osteomyelitis, toxic shock syndrome and pneumoniaS. aureus are still in short supply. In the last years several very useful constructs have been made which aid in the study of S. aureus using fluorescent proteins, using either replicating plasmids2bursa aurealis transposon in the genome of the target strains created for and available through the Nebraska Transposon Mutant LibraryS. aureus.The use of fluorescence reporters is clear in the field of microscopy and flow cytometry analysis. However, also in plate readers with the capability to measure fluorescence both growth (constitutive promoter) as well as specific promoter activity can be measured, such as demonstrated in ref. S. aureus research community23sarA-P1 promoter and the expression cassette is integrated in the genome of the S. aureus strains by homologous recombination. As an intermediate, but similarly useful tool, we produced replicating plasmids containing all mentioned reporter genes, and wherein the sarA-P1 promoter, which is flanked by restriction sites, can be replaced by any other promoter of interest for expression studies. With these constructs it becomes possible to study multiple promoter activities in different fluorescent colours. For example, it is possible to transform a GFP containing plasmid under control of a promoter of your interest, in bacteria constitutively expressing mAmetrine or CFP from its chromosomal location, and analyse promoter activity compared to bacterial growth in conditions where OD cannot be measured reliably due to the turbidity of the medium used or in  microscopy when non-fluorescent cells are very difficult to spot.In this study, we build upon the constructs already available to the E. coli DC10B or E.coli Top10F′. Correct plasmids isolated from E. coli DC10B were directly transformed to S. aureus Newman, MW2 and USA300 strains by electroporation as describedE.coli Top10 F′ it was first introduced in S. aureus RN4220, re-isolated and subsequently introduced in the target S. aureus strain. E. coli was grown in LB broth or agar. S. aureus was grown in Todd-Hewitt agar or broth. When growing in liquid cultures, both E. coli and S. aureus were grown in 50 ml tubes with maximum 10 ml of growth medium, shaken at 300 rpm at 37 °C. Ampicillin concentrations for E. coli were 100 μg/ml and chloramphenicol concentrations for S. aureus were 10 μg/ml. All restriction enzymes were purchased from New England Biolabs and were used according to the manufacturer’s instructions. PCR was performed using Phusion DNA polymerase (Finnzymes) or PWO polymerase (Roche) for cloning, and using Taq-ready mix (Fermentas) for colony checking. Primers were obtained from Integrated DNA Technologies  and sequencing reactions were performed using BigDye Terminator V3.1 (Life Technologies) and analysed at the Central Sequence Facility at UMC Utrecht, or at Macrogen (Macrogen Easy-seq). PCR purifications were performed using a QIAquick PCR Purification Kit (Qiagen). Ligations were performed using a T4 DNA Ligase (Roche Applied Science) according to the manufacturer’s instructions. Initial fluorescence expression was analysed using an ImageQuant LAS4010  or Leica TSC SP5 inverted microscope equipped with a HCX PL APO 40/0.85 objective (Leica Microsystems), with the correct excitation and emissions spectra for the different fluorescent colours  and S. aureus MW2 genome sequence as a reference for codon usage. A G-block of 750 bp containing the newly designed coding sequence with a GC-content of 29% and upstream RBS and spacing to start of the reporter gene identical to pCM29 was ordered from IDT . Additional restriction sites were added by PCR , mKate2, mCherry, YFP (venus) and CFP (Cerulean) coding genes were amplified by PCR from several templates , checked for correct ligation and absence of mutations by restriction analysis and sequencing.To create the mAmetrine, CFP, YFP, and DsRED expressing vectors, the pCM29 plasmid and PCR amplified reporter DNA were digested with KpnI and EcoRI. To create the mKate2 and mCherry constructs, pCM29 and PCR amplified reporter DNA were digested XmaI-EcoRI. After digestion, pCM29 was dephosphorylated, separated on an agarose gel, and the plasmid backbone was isolated from gel. The digested reporters were ligated into pCM29-backbone. The resulting plasmids were named pTH1 (DsRED), pTH2 (CFP), pTH3 (YFP), pRN10 (mKate2), pRN11 (mCherry), pRN12 (mAmetrine). All plasmids were transformed to competent S. aureus Newman DNA (GenBank: AP009351.1) consisting of the region spanning from the nucleotides 40813-43668 containing the 3′ part of NWMN_0029 and the 3′ part of NWMN_0030 (NWMN29-30) was amplified using primers NWMN_0029-FW_EcoRI and NWMN_0030-RV_KpnI  were checked by sequencing, and were designated pTH100 (sGFP); pTH101 (DsRed); pTH102 (CFP); pTH103 (YFP); pRN110 (mKate); pRN111 (mCherry) and pRN112 (mAmetrine). As an example, a map of plasmid pRN112 is given in The fluorescent reporter expression cassettes present in the replicating shuttle vectors described above were amplified by PCR, and simultaneously a transcription terminator was added. The sequence encoding the transcription terminator was obtained from vector pMUTIN2S. aureus chromosome between genes NWMN_0029 and NWMN_0030  and re-streaked on TH+Cm plates and grown at 45 °C overnight. Single colonies were picked to Todd-Hewitt medium without antibiotics and incubated at 30 °C, 250 rpm overnight. Cultures were diluted 1:1000, cultured during 7 hours (during day time), re-diluted, grown for 16 hours (overnight) etc., resulting in 5–7 dilution-culturing cycles, to allow for double cross over events to occur. The final culture was plated on Todd-Hewitt agar containing 100ng/ml anhydrotetracycline and incubated O/N at 37 °C to select for double cross over mutants. The anhydrotetracyclin induces the expression of the counter-selection marker present on the pJB38 derivative plasmidsThe fluorescent protein expression cassettes were integrated markerless into the 660 of 0.01 and 150 μl was transferred to selected wells of a clear 96 well flat bottom polystyrene tissue culture plate (Greiner). The plate was incubated directly in a Fluostar Omega or Clariostar plate reader (BMG labtech) at 37 °C with constant double orbital shaking (400 rpm) in between measurements. Both the optical density at 660 nm and fluorescence of mAmetrine, CFP, GFP, YFP DsRED, mKate, mCherry were measured every 10 minutes for each well. The signal from 4 identical wells was averaged and corrected for blank wells containing only medium. Settings for the wavelengths measured using the Fluostar and Clariostar plate readers are given in Bacterial growth  and fluorescence were measured. Bacteria were diluted to an ODS. aureus USA300 containing plasmid pRN12 were loaded in the wells of a flat bottom 96 well plate. Excitation was scanned using settings: excitation scan: 339.0–484.0; stepwidth 1.0 nm, bandwidth 10 nm; emission wavelength at 519 nm, bandwidth 16 nm. Emission was scanned using settings: excitation wavelength 401 nm, bandwidth 16 nm; Emission scan 447.0–624.0 nm; stepwidth 1.0 nm, bandwidth 8 nm. To correct for autofluorescence, a similar overnight culture of wildtype S. aureus USA300 was taken along, and the spectra from this scan were used as blank.The excitation and emission spectra were recorded using a BMG labtech Clariostar plate reader. 100 μl of an overnight culture of Microscopic image acquisition of fluorescent bacteria was performed using a Leica TSC SP5 inverted microscope equipped with a HCX PL APO 40 × 0.85 CORR CS and HCX PL APO 63x/1.40–0.60 oil immersion . The microscope was encased in a dark environment chamber with temperature control. The cells and bacteria were monitored in brightfield, using wide field epifluorescence, or in confocal mode setting. Using the correct settings, it was possible to obtain an image where four fluorophores could be separately identified in one sample. The separate channels were combined using Leica LAS AF software.E. coli and S. aureus and contains sGFP under the control of sarA-P1 promotersarA-P1 promoter, allowing for easy exchange of this promoter with any other promoter to study its expression. In their present form with the sarA-P1 promoter these plasmids give a very strong fluorescent signal, and it is possible to precisely separate the cells expressing each of the four different fluorescent proteins in one sample . There is 666 bp spacing between the two open reading frames, and the presence of an endogenous EcoRV recognition site in the region between genes 29 and 30  in Newman which was not present in the cloning vector pJB38 was of practical benefit , present in strains MSSA476 and MW2. In the later strain integration was not more problematic than with strain Newman. Note that in the different S. aureus strains the gene names and numbers differ. For USA300 the genes are not designated gene numbers 29 and 30, instead gene NWMN_0029 is identical to SAUSA300_0087 (due to the integration of SSC-mec and ACME upstream of the selected site). S. aureus strains.Finally, the region chosen was highly conserved in the sequenced S. aureus strain Newman another region showed to be adhering the criteria set above, and could be useful for making double labelled strains or stable integration of other genes of interest. This is the genomic region between genes NWMN_0048/NWMN_RS00280 and NWMN_0049/NWMN_RS00285 (63771 bp–64264 bp). The reason not to select this region here was because it is slightly less conserved across most S. aureus strains compared to the 29_30 region. It was 100% homologous to strains Newman, Col, USA300, NCTC 8325, there was a little gap in this region in strains MSSA476 and MW2, and a homology below 95% in N315, Mu50.When going through the genome of S. aureus strains, were re-annotated at NCBI, and their locus tags have changed14Recently all bacterial genomes, including those of most S. aureus, which can be used for chromosomal integrationS. aureus genome. Markerless insertion of the constructed fluorescent expression cassettes mAmetrine, CFP, sGFP, YFP, mCherry, mKate was efficient, both in stains Newman, MW2 and USA300. Furthermore, the sGFP cassette (pTH100) was successfully inserted in strain SH1000 . Unfortunately, the DsRED construct was difficult to obtain, and when a clone with the construct integrated in the chromosome was eventually isolated it rapidly lost DsRED expression, likely due to secondary mutations. This again indicated, as seen with the replicative plasmid, that DsRED was a problematic fluorescent protein to express in the S. aureus strains used. As an alternative we have created both mCherry and mKate constructs, whose excitation and emission spectra are both at a longer wavelength compared to DsRed, allowing for improved separation of multiple fluorophores in the same sample.pJB38 is a plasmid with a temperature sensitive replicon for S. aureus, we have grown both the WT strain and the reporter containing strains in a microplate reader. As is clear from 660 and fluorescence measurements, with the most similarity between optical density and fluorescence signal for GFP, CFP and YFP. The mAmetrine signal also followed the growth curve, but the mKate and mCherry signals are lagging, likely due to lower folding kinetics of these fluorescent proteins660 obviously plateaus, but the fluorescence signal continues to accumulate, indicating that the activity of the SarA-P1 promoter continues into stationary phase. Our results demonstrate that for the constructed reporter strains measuring fluorescence is a suitable alternative to measuring OD if turbidity due to the presence of eukaryotic cells such as neutrophils will prevent correct OD measurements. Depending on the type of fluorescent protein chosen the signal should be corrected based on the known maturation characteristics of the protein.To test whether the newly integrated reporters influenced the growth of S. aureus, which will greatly enhance research on this harmful bacterium. Alternatively, in a co-infection experiment, one could use different fluorescent chromosomal encoded markers in each strain, which after the experiment can readily be quantified using flow cytometry or, after plating, simply counting the number of blue, green and red fluorescent colonies using a fluorescence imager such as a LAS4010.The constructs described here have a broad spectrum of applications in biological research. By using different fluorescent proteins for the specific promoter activity and the genomic integrated reporter, you can differentiate between bacterial growth and promoter activity. This setup will give novel information in a quick and dynamic way. This can be done with any promoter of S. aureus. We have tested Bacillus subtilis and Paenibacillus sp. Both organisms become brightly fluorescent when transformed with the pCM29 plasmid (data not shown), demonstrating that the collection of replicative plasmids described here is also able to drive high expression of fluorescent proteins from the sarA-P1 promoter in other Firmicutes.The plasmid backbone used for all reporter expression plasmids (from pCM29) contains the open reading frame encoding the Rep-1 protein for rolling circle replication in gram-positive hostsHow to cite this article: de Jong, N. W. M. et al. Fluorescent reporters for markerless genomic integration in Staphylococcus aureus. Sci. Rep.7, 43889; doi: 10.1038/srep43889 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The study aimed to evaluate the influence of socioeconomic factors on rheumatoid arthritis (RA) patients' access to biologics in Romania. Cross-sectional data were collected in January 2014 from the Romanian Registry of Rheumatic Diseases (RRRD) comprising all RA patients on biologics from 42 Romanian counties. “Territorial” access to biologics was defined by patients receiving biologics in their home county. A county was “equitable” if <25% of RA patients received biologics outside it.p < 0.001). Gross domestic product (GDP) in 1000 €/capita/county (odds ratio (OR) = 1.224) and number of physicians/1000 inhabitants/county (OR = 2.198) predict territorial access to biologics and also predict the number of territorially treated RA patients. Inequitable counties exhibited significantly lower socioeconomic indicators than equitable counties. The RRRD included 4507 RA patients aged 56.7 ± 12.1 years, with a disease duration of 12.1 ± 8.3 years. Urban dwellers (67.8%) had a significantly higher prevalence of territorial biologic access than rural dwellers (83.1% compared to 74.1%;  In Romania, RA patients' access to biologics varies significantly between counties. Urban dwellers and patients living in counties/regions with high living standards are more likely to receive biologics locally than those living in more deprived areas. Rheumatoid arthritis (RA) is a systemic inflammatory autoimmune disorder affecting approximately 1% of the general population . RA can Since RA usually affects professionally active people, it has important economic consequences, adding costs to patients and their families and to society through extensive use of health resources and loss of work productivity , 5. The In order to be eligible for bDMARDs in Romania, RA patients must be nonresponders to two different conventional synthetic DMARDs (csDMARDs) just like in other European countries, but they must fulfill other severity criteria which are stricter . While sIn order to be considered for bDMARDs therapy, Romanian RA patients must fulfill four criteria: (a) the diagnosis of RA to be be made by a rheumatologist and it should fulfill the 2010 RA classification criteria ; (b) eitRA activity is assessed using the composite 28-joint count disease activity score (DAS28), which is based on the number of tender joints, number of swollen joints, ESR or CRP, and the visual analog scale for patient-reported general health : patientThe mentioned poor prognosis factors include age under 45 years; rheumatoid factor and/or anticitrullinated protein antibodies 10 times the ULN; ESR > 50 mm/h or CRP > 5 times the ULN; erosions on X-rays, ultrasound or magnetic resonance imaging; Health Assessment Questionnaire score above 1.5; extra-articular manifestations.As soon as a patient fulfills the above criteria for bDMARDs use, the attending rheumatologist uploads the data into the RRRD, which is a national electronic database comprising all RA patients treated with biologics in Romania. Prior to treatment and inclusion in the RRRD, all patients signed an informed consent form for both bDMARD therapy and scientific use of their data.For the purpose of this study, the variables were collected in January 2014 using a cross-sectional study design: demographics  and RA-specific variables . The geographical distribution of patients  was reconfigured from a treatment perspective: if the patient received the bDMARD in home county, the case was considered “territorial” access; if the patient received the bDMARD outside home county, the case was considered “extraterritorial” access. Similarly, if a county had less than 25% of its RA patients treated outside, the county was considered to have “equitable” access; if more than 25% of its RA patients were treated outside, the county was considered to have “inequitable” access. The local and RRRD ethics committees approved the study protocol.Socioeconomic indicators were collected from the National Institute of Statistics Yearbook (EUROSTAT ) and incThe normal distribution of the data was assessed using descriptive statistics, normality and stem-and-leaf plots, and the Lilliefors corrected Kolmogorov-Smirnov test. The age of the patients and their disease duration were distributed normally and therefore they were reported as “mean ± standard deviation.”χ2 test with a Cramer's V statistic for effect size. The differences between continuous variables  between patients with territorial or extraterritorial biologic access were assessed using t-tests. Effect size for these t-tests was calculated by Cohen's d statistic, approximated by running the t-tests using the standardized values (Z scores) of the independent variables and observing the mean difference output of these t-tests. A binary logistic regression model was computed in order to predict the likelihood that RA patients will have territorial bDMARD access using the following predictors: age (years), area of residence , GDP (expressed in 1000 €/capita/county) and number of physicians/1000 inhabitants/county. The results were reported in terms of odds ratios (OR) with 95% confidence intervals (CI).On a patient-based analysis (4507 patients), the association between residence  and bDMARD access  was assessed using a On a county-based analysis (42 counties), GDP/capita, number of physicians/1000 inhabitants, and number of rheumatologists/county exhibited a nonnormal distribution; therefore the differences of these scale variables according to type of county (equitable and inequitable) were studied using Mann–Whitney tests . For the same reason, the correlations between these scale variables among themselves and with the number of territorially and extraterritorially treated RA patients per county were computed using Spearman's rho coefficients. Two hierarchical multiple linear regression models were constructed in order to predict the number of RA patients with territorial access to biologics. Both models used GDP/capita as predictor in the first step. For the second step, in the first model the number of physicians/1000 inhabitants was added as predictor, while in the second model the number of rheumatologists/county was added as predictor. These predictors were previously normalized by extracting square roots (number of rheumatologists/county) or by calculation of their natural logarithms (number of physicians/1000 inhabitants). Since the number of physicians and the number of rheumatologists are not independent variables (the number of physicians includes the number of rheumatologists), they were not included together in the same regression model.p < 0.05. All the statistical analysis was done using IBM SPSS Statistics version 22.0 for Windows .The statistical tests were considered significant if Until January 2014, the RRRD included 4507 patients with RA: 4267 (94.7%) treated with biologics and 240 (5.3%) with approved biological therapy, but treatment not yet started. The patients had a mean age of 56.7 ± 12.1 years and a mean RA duration of 12.1 ± 8.3 years. The majority of patients were women (3842 patients—85.2%) and the majority of patients lived in urban areas (3056 patients—67.8%). In the sample, 80.2% (3614) of RA patients benefited from territorial bDMARD access, while 19.8% (893) had extraterritorial bDMARD access , Table 1Compared to RA patients with territorial bDMARD access, those with extraterritorial bDMARD access had equivalent mean ages but came from counties with significantly lower socioeconomic indicators .χ2(5) = 1018.9; p < 0.001) explained 32.1% of bDMARD accessibility (Nagelkerke R2 = 0.321) and it correctly classified 81.7% of patients. Accounting for all other predictors included/present in the model, GDP expressed in 1000 €/capita/county  and number of physicians/1000 inhabitants per county  significantly increased the likelihood that patients will have (equitable) territorial bDMARD access.The logistic regression model : 53.7% (805/1498) of patients treated in Bucharest come from a different county. Compared to equitable counties regarding bDMARD treatment, the inequitable counties exhibited significantly lower socioeconomic indicators .p < 0.001; rho = 0.448, p = 0.003; rho = 0.337, p = 0.034, resp.), and they were significantly and negatively correlated with the number of extraterritorially treated RA patients/county .The number of rheumatologists/county, the number of physicians/1000 inhabitants/county, and GDP/capita were significantly and positively correlated with the number of territorially treated RA patients/county , the number of physicians/1000 inhabitants/county , and GDP/capita .p < 0.001) and with the number of rheumatologists/county .Two hierarchical regression analysis models were computed to predict the number of territorially treated RA patients using GDP (1000 €/capita/county) in the first step and then adding either the number of physicians/1000 inhabitants/county or the number of rheumatologists/county as predictors . GDP on The aim of the study was to assess the impact of socioeconomic factors on Romanian RA patients' accessibility to bDMARDs. In this sense, we found that rural habitat and poor county socioeconomic indicators  are associated with lower access to bDMARDs. In order to discuss the relevance of these results, we must first review the characteristics of the sample. Compared to patients from other European RA registries , RA patiObservational studies have reported that financial factors such as macroeconomic conditions, income, and national health expenditure are major influencing factors of bDMARD accessibility in European countries , 20–23. p < 0.001). While other more economically developed countries are also facing a shortage of rheumatologists [The observed predictive power of the number of physicians/rheumatologists for bDMARD access of RA patients is intrinsically linked to the characteristics of the disease and the structure of the health system. In this sense, the number of physicians has been long used as an indicator of socioeconomic development. Nationwide, for a population of roughly twenty million inhabitants, there were 235 active rheumatologists. Out of the 42 administrative divisions of Romania, there were 7 counties without a rheumatologist, while in the capital there were 75 active rheumatologists. The distribution of rheumatologists among the 41 counties and the capital was uneven and it was correlated with territorial economic level, as measured by the GDP/county   and the In Romania, accessibility of RA patients to biological therapy varies significantly between different counties. Areas with low socioeconomic level do not offer equal and fair therapeutic opportunities for RA patients compared to other national areas: patients with RA living in urban areas and counties/regions with high living standards are more likely to receive biological agents locally than those living in more deprived areas. Studies investigating patient-reported barriers to biologic therapy are needed in the Romanian population.


Use of nitarsone, an arsenic-based poultry drug, may result in dietary exposures to inorganic arsenic (iAs) and other arsenic species. Nitarsone was withdrawn from the U.S. market in 2015, but its use in other countries may continue.We characterized the impact of nitarsone use on arsenic species in turkey meat and arsenic exposures among turkey consumers, and we estimated cancer risk increases from consuming turkey treated with nitarsone before its 2015 U.S. withdrawal.Turkey from three cities was analyzed for total arsenic, iAs, methylarsonate (MA), dimethylarsinate, and nitarsone, which were compared across label type and month of purchase. Turkey consumption was estimated from NHANES data to estimate daily arsenic exposures for adults and children 4–30 months of age and cancer risks among adult consumers.Turkey meat from conventional producers not prohibiting nitarsone use showed increased mean levels of iAs (0.64 μg/kg) and MA (5.27 μg/kg) compared with antibiotic-free and organic meat  and meat from conventional producers prohibiting nitarsone use . Samples with measurable nitarsone had the highest mean iAs and MA . Nitarsone was higher in October samples than in March samples, possibly resulting from increased summer use. Based on mean iAs concentrations in samples from conventional producers with no known policy versus policies prohibiting nitarsone, estimated lifetime daily consumption by an 80-kg adult, and a recently proposed cancer slope factor, we estimated that use of nitarsone by all turkey producers would result in 3.1 additional cases of bladder or lung cancer per 1,000,000 consumers.Nitarsone use can expose turkey consumers to iAs and MA. The results of our study support the U.S. Food and Drug Administration’s removal of nitarsone from the U.S. market and further support its removal from the global marketplace.http://dx.doi.org/10.1289/EHP225Nachman KE, Love DC, Baron PA, Nigra AE, Murko M, Raber G, Francesconi KA, Navas-Acien A. 2017. Nitarsone, inorganic arsenic, and other arsenic species in turkey meat: exposure and risk assessment based on a 2014 U.S. market basket sample. Environ Health Perspect 125:363–369;  The appEvidence shows that the use of arsenic-based drugs in food animal production results in human dietary exposures to arsenic, including inorganic arsenic (iAs) , 2016, aHistomonas meleagridis , we estimated the lifetime average daily exposure to these arsenic species among turkey consumers and estimated cancer risks associated with ingestion of iAs attributable to arsenical drug use in animal production.A total of 184 turkey samples were included in our study, including products from 14 producers representing 64% of the 2014 U.S. turkey market . Turkeysn = 56) were purchased in Baltimore, Maryland; 10 additional samples were purchased in October  to increase the study sample size for that month. There was slight variation in grocery chains and stores visited in March and October, but overall, there was considerable overlap in purchase sites.Samples purchased in March 2014 were purchased from retail grocers in three geographically diverse U.S. metropolitan areas , selected to cover the East Coast, the West Coast, and the Midwest. We visited 12 unique stores (9 supermarket chains) in Los Angeles, 10 unique stores (6 supermarket chains) in Denver, and 12 unique stores (10 supermarket chains) in Baltimore, for a total of 36 unique stores representing 23 supermarket chains. All samples collected in October 2014 (All samples were taken from packages of raw turkey meat (ground or whole cuts). Within each store, we purchased two packages of a single type (ground or whole cuts) of turkey for each brand available (multiple brands were purchased when available within a store), including store-branded products. We also purchased conventionally produced products and U.S. Department of Agriculture (USDA) Organic and antibiotic-free–labeled turkey products of each type and brand, when possible. If only one package was available for a type and brand of turkey, then one single package was purchased. For example, from a given store in one of the study cities, based on available products, we purchased two packages of conventional ground turkey and two packages of conventional whole cut turkey from Producer B and two packages of conventional ground turkey from Producer A. In this particular store, neither antibiotic-free nor USDA Organic turkey products were available.The mean [standard deviation (SD)] meat weight in the purchased package was 0.52 (0.15) kg for ground meat and 0.69 (0.28) kg for whole cuts. From each package, 60- to 70-g aliquots of meat were collected and processed as described below for arsenic analyses. All whole pieces of meat examined were exclusively breast tissue. Ground meat, however, was often a mixture of breast and other unspecified muscle tissue. We did not purchase cooked products, deli meats, or products such as hot dogs, sausages, or frozen meals containing turkey because mixed ingredients in the packages could interfere with sample analysis. For example, some packages of turkey deli meat contain rice starch or carrageenan , which are used for gelling and thickening and could have been a source of added arsenic.Samples were prepared for freeze-drying as previously described . Brieflyn = 39) over the course of the study. The turkey samples were analyzed in duplicate for their total arsenic content; when duplicate values differed by > 10%, which occurred with only 4 of the 184 samples, the sample was re-analyzed. Taken together, 432 total arsenic measurements  were made.Detailed descriptions of laboratory reagents, standards, and reference materials; instrumentation; extraction of arsenic species; and HPLC-ICPMS analyses are provided in “Detailed Laboratory Methods Section” in the Supplemental Material. In brief, the total arsenic contents of the freeze-dried turkey samples were determined using inductively coupled plasma mass spectrometry (ICPMS–Agilent 7900 ICPMS from Agilent Technologies) following microwave-assisted acid mineralization in an UltraCLAVE III microwave system (MLS GmbH). Arsenic standards in the range 0.01–20 μg As/L were used for external calibration; germanium  was added to standards and samples to normalize matrix effects. Because there is currently no turkey or chicken meat reference material certified for total arsenic content, we used the standard reference material ERM-BC211 rice  for qualn = 9). Detection limits, on a dry weight basis, were 1, 1–2, 1, 1, and 1 μg/kg for total arsenic, iAs, MA, DMA, and nitarsone, respectively.Arsenic species were determined in duplicate analysis in alkaline aqueous extracts of the freeze-dried turkey samples by using anion-exchange high performance liquid chromatography (HPLC) coupled to an ICPMS which served as an arsenic-selective detector. When the values for arsenic species in the duplicates differed by > 20%, the sample was re-analyzed. Separation of dimethylarsinate (DMA), methylarsonate (MA), iAs, and nitarsone was achieved by gradient elution using ammonium carbonate buffer with a Dionex AS15A anion exchange column. For quality control, we used the standard reference material ERM-BC211 ; we obtained 116 ± 4 μg As/kg for DMA and 99 ± 4 μg As/kg for iAs . For total arsenic, iAs, MA, and DMA measurements below the detection limit, we imputed the value of the detection limit divided by the square root of 2. For nitarsone, samples below the detection limit were given the value of 0. For each arsenic species, wet weight sample concentrations were calculated for each sample by multiplying its dry weight concentration by the sample-specific water loss dilution factor.Samples were analyzed in a random order, and the lab was blinded to sample identity. Thirty blinded duplicate samples were analyzed separately to evaluate the performance of the method but were not included as additional samples in the main analysis.We categorized samples into two groups based on their package labels to compare USDA Organic or antibiotic-free to conventionally produced samples. It is critical to note that although the USDA Organic certification program explicitly prohibits the use of arsenic-based pharmaceuticals in animal production , the labStatistical analyses were performed with Stata 14 (StataCorp LLC). Mean arsenic species concentrations and 95% confidence intervals (CIs) were calculated to evaluate differences among categories of turkey samples . The seasonal analysis was also restricted to Baltimore-only samples in a sensitivity analysis. Pearson’s coefficients were used to assess correlations between concentrations of total arsenic and arsenic species including nitarsone. Statistical significance was two-tailed and was set at α = 0.05.Intake rate calculation. Population intake rates for turkey meat were derived from the dietary recall component of the 2003–2010 cycles of the National Health and Nutritional Examination Survey (NHANES)  × IR)/BW, [1]where LADD is the lifetime average daily dose (in milligrams per kilogram body weight per day), [As] is the mean arsenic species concentration (in milligrams per kilogram) in the specific category of turkey product, IR is the per capita turkey intake rate, and BW is body weight (80 kg BW) .We also estimated average daily doses of arsenic species for children between the ages of 4 and 30 months who consume baby food made from turkey using the formula:ADD = ([As] × IR)/BW, [2]where ADD is the average daily dose (in milligrams per kilogram body weight per day), [As] is the mean arsenic species concentration (in milligrams per kilogram), IR is the turkey intake rate (0.019 kg/day), and BW is body weight (11.01 kg BW).For iAs, we also estimated the difference in iAs intake for consumers of turkey produced with nitarsone compared with consumers of turkey produced without nitarsone by subtracting the mean iAs concentration for the antibiotic-free or USDA-certified Organic group from the mean iAs concentration for the “conventional with no known arsenical policy” group. The resulting value is an estimate of the added iAs in the meat from the use of nitarsone. The calculation of the LADD and the risk using this value reflects an estimation of the added iAs exposure and excess lifetime cancer risk attributable to the decision to use nitarsone in turkey production. We then estimated cancer risk in turkey consumers by multiplying this LADD by the U.S. EPA Integrated Risk Information System (IRIS) cancer slope factor (q*) for iAs:iAs × q*. [3]Risk = LADDThe U.S. EPA IRIS toxicological review of inorganic arsenic is currently under reassessment and review by the National Research Council; consequently, we used a value of 25.7/[mg/kg BW/day] proposed in a draft version of the toxicological review in 2010, reflecting the U.S. EPA’s most recently published analysis of the epidemiologic literature and corresponding to lung and bladder cancers . ToxicitWe conducted sensitivity analyses to examine how the relative market share of each turkey producer would affect the estimated population cancer burden attributable to turkey consumption in the United States if nitarsone had not been withdrawn from the market. Using the most recent (2014) producer-specific data (see Table S1) from the A second approach was considered to allow for comparisons between producers who likely use nitarsone and those who do not. Considering only turkey producers for whom we have samples (representing 64% of the U.S. turkey market), we used the 2014 producer-specific data from the National Turkey Federation to calculate the relative market share for each producer by dividing its 2014 production quantity by the production quantity summed across the producers included in our study. We then categorized producers into a nitarsone-positive group (including producers with at least one sample with measurable nitarsone) and a nitarsone-negative group (including producers with no measurable nitarsone in any sample). Then, weighted mean iAs concentrations were calculated for the nitarsone-positive (0.74 μg/kg) and nitarsone-negative (0.11 μg/kg) groups separately. The difference in those weighted means (0.63 μg/kg) was considered the iAs attributable to nitarsone use. This value was then used in the previously described models to estimate LADD, risk, and annual population cancer burden.The mean total arsenic concentration in turkey meat was 11.2  μg/kg . Mean iAp = 0.026), iAs (p = 0.041), and MA (p = 0.036) concentrations than the combined USDA Organic and antibiotic-free labeled samples. Measurable nitarsone was observed in 21% of conventional samples, compared with 10% nitarsone in the combined antibiotic-free and USDA Organic samples. Within the conventional group, further differences were observed when samples were grouped by whether producers had a known policy or no known policy prohibiting arsenic use. Samples from conventional producers with no known policy (n = 87 samples) had significantly higher mean concentrations for all arsenic species than samples from conventional producers with prohibitory policies . Nitarsone was not found in samples from conventional producers with prohibitory policies but was detected in 28% of samples from producers without known policies.By package label, conventionally produced samples  had higher total arsenic  (p < 0.001).The greatest differences in arsenic species concentrations were observed between samples with and without measurable nitarsone (arisons) .The largp = 0.009) between samples purchased in March [0.40 ] and in October [0.79 ]. Nitarsone concentrations were 0.65  μg/kg for samples purchased in October and 0.11  μg/kg for samples purchased in March (p = 0.07). Differences were not observed for the other species , although the mean total arsenic and MA concentrations were higher for samples purchased in October.Seasonal comparisons were examined using only Baltimore samples. A significant difference in iAs (in micrograms per kilogram) was observed (n = 8) in the “other” category had measurable nitarsone. Among producers with measurable nitarsone, mean values ranged from 0.05 to 1.09 μg/kg.Nitarsone residues were measured in at least one sample from 7 of the 19 producers included in the study (producers with three or fewer samples were grouped into an “other” category); among those, the frequency of nitarsone detection varied considerably, from 7% to 100%. The producer with a rate of 100% nitarsone measurement was part of the “other” category and accounted for two samples; none of the remaining producers (We estimated Pearson correlation coefficients between arsenic species (see Table S2). Moderate to strong correlations were observed for all measured arsenic species, with the weakest correlation being between iAs and DMA (0.54).Estimates for LADDs of arsenic species by sample characteristics are presented in –6 mg/kg BW/day, compared with 4.5 × 10–7 mg/kg BW/day for adults).By group, iAs exposures were higher among samples from conventional producers without policies prohibiting nitarsone use and samples with measurable nitarsone and lower among samples from the combined antibiotic-free and USDA Organic group and conventional producers where use was prohibited by company policy. Similar patterns were seen for average daily doses for children . One not–7 mg/kg BW/day. Based on the U.S. EPA’s proposed cancer slope factor for iAs of 25.7/(mg/kg BW/day)  would ingest an additional 0.01 μg iAs per day, resulting in a LADD of 1.22 × 10 BW/day) , this li BW/day) , 24% of –7 mg/kg BW/day, a risk of 9.07 × 10–6, and an annual population cancer burden of 10 cases per year attributable to turkey consumption in the United States (including contributions from both nitarsone users and nonusers). To refine this estimate of burden, we estimated the market share–adjusted population cancer burden from iAs attributable to nitarsone use by separating producers into nitarsone use and nonuse categories based upon our data . Under this scenario, we estimated a LADD of 3.09 × 10–7 mg/kg BW/day, a risk of 7.93 × 10–6, and an annual population cancer burden of 9 cases per year. Burden estimates under these two modeled scenarios were higher than the original burden estimation, which did not consider producer market share. The reason for this difference is the uneven market share across producers; for example, the producer with the largest market share was a nitarsone user with the highest mean iAs concentration.We conducted additional sensitivity analyses to consider the influence of the market share of turkey producers on cancer burden estimates had nitarsone not been withdrawn. Using 2014 producer-specific data from the Because cancer slope factors are not available for other arsenic species, the contributions of MA and DMA to lung and bladder cancer risks cannot be estimated despite evidence supporting their potential carcinogenicity .Before their withdrawals in 2014 and 2015 , arsenicOur results come soon after the FDA’s withdrawal of the marketing approval of nitarsone in the United States . The basThe FDA action against arsenical drugs in the United States does not affect their use in other countries, nor does it prevent U.S.-based pharmaceutical companies from selling these drugs in other countries. For example, arsenicals are approved for use in China and can be found in commercial feed for poultry and swine and in animal manure in China . The USDOur assessment did not specifically address noncancer health concerns linked to chronic iAs exposure , and litWe were unable to account for other potential sources of arsenic that could be related to differences in the impact of nitarsone use on measured arsenic species, including arsenic in the drinking water used by turkeys or nonarsenical drug–related sources of arsenic in the feed used for turkeys.Future work  is needed to contextualize other aspects of the current and historic health burden imposed on turkey consumers by the use of nitarsone.Our study provides evidence that the use of nitarsone in turkey production can contribute to exposure to iAs and methylated arsenic species among turkey consumers. Our findings support the FDA’s precautionary decision to withdraw marketing approval for nitarsone in 2015, and they support similar actions by other governments and international agencies to protect public health in all populations.(159 KB) PDFClick here for additional data file.


Risk scoring systems are used to evaluate patients with upper gastrointestinal bleeding (UGIB). We compared Glasgow-Blatchford score (GBS), modified GBS (mGBS), and Pre-endoscopy Rockall score (Pre-E RS) for immediate application without endoscopic findings in predicting the need of interventions and the 30-day mortality in patients with UGIB.Patients who visited the emergency room with UGIB from January 2007 to June 2016 were included. GBS, mGBS, and Pre-E RS were obtained for all patients. The area under the receiver-operating characteristic curves (AUC) was used to assess the accuracy of the scoring systems to determine the need for interventions and 30-day mortality. Also, we investigated the potential cutoff scores for predicting 30-day mortality and the need for interventions.P < 0.0001). In predicting 30-day mortality, Pre-E RS (AUC = 0.929) outperformed GBS  and mGBS . Based on AUC analyses of sensitivities and specificities, the optimal cutoff mGBS and GBS for the need for interventions was 9  and 9  respectively, and optimal cutoff Pre-E RS for 30-day mortality was 4 .In predicting the need for interventions, GBS (AUC = 0.727) and mGBS (AUC = 0.733) outperformed Pre-E RS (AUC = 0.564, GBS and mGBS are considered to be moderately accurate in making an early decision about the need of interventions in patients with UGIB. Pre-E RS is considered to be highly accurate in early detection of patients at high risk for 30-day mortality without endoscopic findings. In addition, we suggested potential cutoff scores to predict the need of interventions for GBS and mGBS, and 30-day mortality for Pre-E RS. Further studies are needed to confirm the clinical applicability of results. Upper gastrointestinal bleeding (UGIB), a common medical emergency, is reported in the emergency room by 5% per year and accounts for 2~3% of hospitalization in developed countries . MortaliThe 2010 International consensus guidelines recommended early risk stratification using proven factors for the management of patients with UGIB . The recSeveral scoring systems have been developed to evaluate patients with UGIB , 8. DeciIn this study, we determined the immediate applicability without endoscopic findings of the GBS, mGBS, and Pre-E RS systems in estimating the need for interventions and predicting the 30-day mortality in patients with UGIB. Also, we investigated the potential cutoff scores to predict the need of interventions and 30-day mortality in applicable scoring systems.This is the retrospective study which was carried out at a single teaching center. The medical records were used to recognize patients who visited the emergency department with UGIB between January 2007 and June 2016.Patients >18 years of age who visited the emergency department with symptoms and signs of UGIB from either variceal or nonvariceal source were involved in the study. Patients diagnosed with lower gastrointestinal bleeding were excluded. Patients stable enough for direct discharge from the emergency room had been recommended out-patient endoscopic examination within 2 days according to protocol; and patients who had not visited the hospital after discharge were excluded. All participations were informed of the study purpose and written consents were obtained following the requirements of the Institutional Review Board of Kyung Hee University Hospital at Gangdong. This study was approved by the Institutional Review Board of Kyung Hee University Hospital at Gangdong (KHNMC IRB 2016–07-026).One junior doctor reviewed each subject’s hospital record including admission notes, discharge records, laboratory data, and endoscopic results to identify the presence of UGIB. Data on age, sex, medication use, drinking, smoking, pulse rate, blood pressure, physical exam findings, laboratory results, co-morbidities, and endoscopic results were collected from each patient. In addition, data concerning interventions including blood transfusion, endoscopic intervention, radiologic intervention, or surgery and 30-day mortality were collected.The high dose proton pump inhibitor  was administered to all subjects with UGIB intravenously. The terlipressin  was administered to subjects who were considered to have variceal bleeding intravenously. Actions were taken for patients requiring interventions. Interventions is defined as medical or surgical procedures including as follows; blood transfusion, combination endoscopic hemostasis using dilute epinephrine injection plus second hemostasis modality , radiologic arterial embolization, or surgery. Endoscopic hemostasis was performed when Endoscopic findings were judged as Forrest classification Ia, Ib, or IIa, and radiologic intervention or surgery were performed when endoscopic hemostasis failed.The GBS, mGBS, and Pre-E RS were calculated for each patient, as previously described Table  7–10]. –10. 7–10p < 0.05.Descriptive analyses were conducted using SPSS . Categorical variables were expressed by number and percentage occurrence frequency. Receiver-operating characteristic (ROC) curves were conducted, and the area under the ROC curve (AUC) with a 95% confidence interval was calculated with MedCalc . Estimates of sensitivity, specificity, positive predictive value, and negative predictive value with 95% confidence intervals (CIs) were obtained for each scoring system, and the ability to predict the need for interventions and the 30-day mortality was compared with MedCalc (version 16.4.3) using the method described by DeLong et al. . The sign = 288, 48.8%), hematemesis , hematochezia , and syncope . 202 patients (34.2%) were taking aspirin, 90 patients (15.3%) were taking warfarin, and 26 patients (4.4%) were taking non-steroidal anti-inflammatory drugs (NSAIDs). 449 patients (76.1%) received blood transfusion. 441 patients (74.8%) underwent gastroscopy within 24 h of presentation to the hospital. Gastroscopy was not performed in 2 patients because they were considered as low risk and unlikely to require endoscopic therapy. Endoscopic intervention and radiologic intervention were performed in 191 (32.4%) and 13 (2.2%) patients respectively, and any surgery to treat UGIB was not performed. The 30-day mortality was 4.2% (n = 25). The averages of GBS, mGBS, and Pre-E RS were 10.0, 9.4, and 2.0, respectively.Table Table p < 0.0001) in predicting the need of interventions. There was not the significant difference between GBS and mGBS in predicting this point. In predicting 30-day mortality, the Pre-E RS (AUC 0.929 [95% CI 0.905–0.948]) outperformed the GBS  and mGBS .Table 2 = 21.675, p < 0.001). The odds ratio for 30 days mortality was 6.753  meaning that the probability of death in the endoscopy performance over 24 h was 6.753 times higher than in the endoscopy performance within 24 h.Table 2 = 19.380, p < 0.001). The odds ratio for 30 days mortality was 6.215  meaning that the probability of death in the endoscopy performance over 24 h was 6.215 times higher than in the endoscopy performance within 24 h. In addition, based on AUC analyses of sensitivities and specificities in our study, the optimal cutoff Pre-E RS to predict 30-day mortality was 4 .Of 372 patients with mGBS >9, 21 patients died in 30 day from presentation. Among them, 12 patients underwent endoscopy after 24 h from presentation, and 9 patients underwent endoscopy within 24 h  was higher compared with previously published studies , 13, becH. pylori infection, contributed to the development of UGIB in coxibs users. The use of non-steroidal anti-inflammatory agents (NSAIDs) and anticoagulation agents associated with aging was considered to increase the development of UGIB by peptic ulcer. In our study, peptic ulcer disease including gastric ulcer and duodenal ulcer was the most common cause of UGIB, and 19.7% of patients took NSAIDs and anticoagulation agents. In our endoscopic findings, 2% of patients showing normal gastroscopic and colonoscopic findings were diagnosed with obscure gastrointestinal (GI) bleeding which is defined as persisting and/or recurrent GI bleeding of unidentified source after negative bidirectional endoscopic evaluation [Lin et al.  reportedaluation . In casealuation .Laine et al.  reportedWhen we planned this study, we considered a research involving only patients with non-variceal bleeding. However patients with symptoms and signs of UGIB who visited emergency department could not be distinguished clearly between variceal bleeding and non-variceal bleeding first. So, we analyzed patients with symptoms and signs of UGIB from either variceal or non-variceal source. However, when we analyzed data involving only patients with non-variceal bleeding, the result was similar with that of this study.Several risk scoring systems have been produced to divide patients with UGIB into high- and low-risk categories. 2010 international consensus for UGIB recommended that the use of prognostic scales was needed for early stratification of patients with UGIB according to the degree of risk . Early ip = 0.81), and outperformed the Pre-E RS  in predicting the need of interventions. Stanley et al. [In this study, we used AUC to evaluate the 3 scoring systems for predicting the need of interventions and 30-day mortality in patients with UGIB. An arbitrary guideline proposed by Swets classifies scoring systems as non-informative (AUC = 0.5), less accurate (0.5 < AUC ≤ 0.7), moderately accurate (0.7 < AUC ≤ 0.9), highly accurate (0.9 < AUC < 1) and perfect tests (AUC = 1) . AUC anay et al.  reportedy et al. . Similary et al. , 22. Rocy et al.  reportedy et al. .A recent retrospective study proposed that early gastroscopy should be performed for high-risk patients with GBS >12 to reduce mortality . Our stuThis is the large-scale retrospective study to propose an appropriate scoring system without endoscopic findings for predicting the need of interventions and 30-day mortality in patients with UGIB. Our results may be useful to physicians in the emergency department for early decisions on interventions and prognosis. However, our study had some limitations. First, this was a single-center study from a teaching hospital, hence, our results cannot be generalized to all patients with the symptoms of UGIB. Second, there are generally some biases affecting the veracity of the retrospective study, such as selection bias, information bias and confounding, although all medical records used in this study were reviewed and entered into a database by one medical doctor for the quality of data. Finally, the rate of high-risk patients presenting with the symptoms of UGIB was high in this study. Therefore, further studies are required for accurate clinical applicability of results.The GBS and mGBS are considered to be moderately accurate with good sensitivity and specificity in making an early decision about the need of interventions in patients with UGIB. Pre-E RS is considered to be highly accurate in early detection of patients at high risk for 30-day mortality without endoscopic findings. In addition, we suggested potential cutoff scores to predict the need of interventions in GBS and mGBS and 30-day mortality in Pre-E RS. These risk scoring systems predicting the need of intervention and/or 30-day mortality can contribute to saving lives and alleviating the severity of a patient’s condition. Especially, these scores may be useful in case of emergency where endoscopy are not available. In this sense, the verification of risk scoring systems through the substantial clinical data conducted in this study may be beneficial to both physicians and patients. However, the perfect score should be applicable in pre- and post-endoscopy. Further studies are needed to confirm the clinical applicability of results.


Acquired demyelinating syndromes (ADS) are immune-mediated demyelinating disorders of the central nervous system in children. A nationwide, multicentre and prospective cohort study was initiated in the Netherlands in 2006, with a reported ADS incidence of 0.66/100,000 per year and MS incidence of 0.15/100,000 per year in the period between 2007 and 2010. In this study, we provide an update on the incidence and the long-term follow-up of ADS in the Netherlands.Children < 18 years with a first attack of demyelination were included consecutively from January 2006 to December 2016. Diagnoses were based on the International Paediatric MS study group consensus criteria. Outcome data were collected by neurological and neuropsychological assessments, and telephone call assessments.Between 2011 and 2016, 55/165 of the ADS patients were diagnosed with MS (33%). This resulted in an increased ADS and MS incidence of 0.80/100,000 per year and 0.26/100,000 per year, respectively. Since 2006 a total of 243 ADS patients have been included. During follow-up , 137 patients were diagnosed with monophasic disease (56%), 89 with MS (37%) and 17 with multiphasic disease other than MS (7%). At least one form of residual deficit including cognitive impairment was observed in 69% of all ADS patients, even in monophasic ADS. An Expanded Disability Status Scale score of ≥ 5.5 was reached in 3/89 MS patients (3%).The reported incidence of ADS in Dutch children has increased since 2010. Residual deficits are common in this group, even in monophasic patients. Therefore, long-term follow-up in ADS patients is warranted. Acquired demyelinating syndromes (ADS) are immune-mediated demyelinating disorders of the central nervous system (CNS) in children , 2. ADS ADS may remain monophasic after the first event. Yet, 15–32% of these children will fulfil the diagnostic criteria for paediatric MS within 5 years after the initial attack , 8. MultIn the Netherlands, a multicentre and prospective study was established in 2006 with national coverage for children with a first demyelinating event. Incidence estimates of paediatric ADS and multiple sclerosis have been reported in our prior work for the period between 2007 and 2010 . HoweverChildren younger than 18 years, residing in the Netherlands, and experiencing a first inflammatory demyelinating event of the CNS in the period from 2006 to 2016 have been included in this study. All patients are participants of the PROUD-kids study (PRedicting the OUtcome of a Demyelinating event in children), a prospective, multicentre and observational cohort study. Paediatric neurologists of the eight Dutch academic hospitals and of ten non-academic hospitals took part in this study and included patients to reach nationwide coverage.Diagnoses were made using the revised criteria proposed by the International Pediatric Multiple Sclerosis Study Group (IPMSSG) . PatientAt inclusion, demographic and clinical information of each patient was gathered. Demographic characteristics consisted for example of ethnic background, date and place of birth and family history on familial autoimmune diseases. Clinical characteristics consisted of presenting symptoms, reported infection or vaccination in the preceding 4 weeks, acute treatment and hospitalization. MRI images, serum and CSF parameters were also reviewed when available for diagnostics or evaluation.If patients were not referred to the paediatric MS centre for follow-up, the follow-up data of the patients were provided by the treating physician  and by interviewing the parents through telephone every 2 years after disease onset.Cognitive impairment (CI) and residual neurological deficits were assessed using the most recent neuropsychological assessment (NPA) performed by a paediatric clinical neuropsychologist, and neurological examination by a paediatric neurologist. NPAs were being performed appropriately for age. During the NPA, at least six of the following cognitive domains were being assessed for the presence of cognitive deficits: behaviour, language, intelligence, attention and concentration, memory, executive control functions and visuospatial abilities. Children were classified as cognitive impaired if at least one of these domains was affected.If data on neurological examination or NPA were not available, a standardized questionnaire was administered asking parents or patients about the presence of sensory complaints, motor deficits , bladder complaints (e.g. urge incontinence), maximum walking distance and cognitive impairment (including negatively affected school performance).The Expanded Disability Status Scale is widely used to express disability of patients with MS diagnosis . EDSS 5.Serum AQP4-ab and MOG-ab were tested with cell-based assays (CBA) provided in a central laboratory as described previously , 22. PatThis study was approved by the Medical Ethical Committees of the Erasmus MC in Rotterdam and the other participating centres. Written informed consent was obtained from parents and also from patients if aged > 12 years at presentation.Demographic data of the general Dutch population were provided by Statistics Netherlands . These dU test. To compare the ethnic background of the patients with the Dutch population we used a Z test, with data provided by Statistics Netherlands [p < 0.05. Missing data were removed from the analyses in all subgroups.Chi-square and when appropriate Kruskal–Wallis tests were used to test differences in demographic and clinical characteristics between the different subtypes. For differences in numerical data between subtypes the ANOVA test was used, and when necessary the Mann–Whitney herlands . ResultsFrom January 1, 2011 to December 31, 2016, 165 ADS patients were reported of which 55 (33%) received an MS diagnosis during FU. In this period, the incidence of ADS was 0.80/100,000 per year, ADEM incidence was 0.23/100,000 per year and MS incidence was 0.26/100,000 per year. An overview of the calculated incidences is shown in Fig. n = 55, 23%; from which 16/55 bilateral ON, 29%), transverse myelitis ; other monofocal clinically isolated syndromes , polyfocal CIS , acute disseminated encephalomyelitis  and NMOSD .From January 1, 2006 to December 31, 2016, 353 patients were eligible. Of these patients, 243 patients with a first demyelinating event were included in the study  and presented more often after a reported preceding infection (p < 0.001). The latter also applied to children who experienced a TM as first event (p = 0.01).Regarding the age of onset, children with ADEM were significantly younger than the other presenting phenotypes (n = 104) and a group > 11 years (n = 139), the female:male ratio differed significantly .The ratio between females and males in all ADS patients did not differ significantly between the presenting phenotypes. When the ADS patients are divided into a group aged < 11 years  [Seventy-eight patients (32%) were of non-Caucasian origin. This proportion was significantly higher than the proportion of children of non-Caucasian origin (17%) in the general paediatric population in the Netherlands (< 0.001) . Most ofp = 0.3).Forty-eight percent of all patients had a positive familial history of autoimmune diseases (first- and second-grade family members). Forty-eight (20%) patients reported the presence of rheumatoid arthritis in their family, 40 (16%) reported thyroid diseases, 35 (14%) the presence of other autoimmune diseases (e.g. Wegener’s disease and Crohn’s disease), 15 (6%) the presence of MS, 14 (6%) diabetes mellitus type 1 and 4 (2%) the presence of optic neuritis (ON). No significant difference was observed between the presenting phenotypes considering the familial history (p < 0.001).In all ADS patients, disease onset in winter was most prevalent (32%), compared to spring (28%), summer (22%) and autumn (17%). MOG antibodies were found to be positive in 31 of the 146 tested patients (21%). When comparing the presenting phenotypes, MOG antibodies were most frequently found in patients who presented with ADEM and NMOSD  ON , TM , CIS , polyfocal CIS , and monophasic NMOSD . Of these NMOSD patients, three were tested seropositive for AQP4-ab and four were seropositive for MOG-ab. Seven monophasic patients received chronic immunosuppressive therapy: this was initiated in all AQP4-ab-positive patients, and in one of the MOG-ab-positive patients due to the disease severity at onset , including ADEM , with more girls in the older MS group.Eighty-nine patients were diagnosed with MS in our cohort (37%), of which 87 received the diagnosis within 5 years of follow-up. Of the 89 MS patients, 70 individuals developed a second attack during follow-up, and were thus diagnosed with clinically definite MS (CDMS). In these patients, the median time to CDMS was 9 months (IQR 4–27). After 2 years of follow-up, 74 percent of the MS patients developed CDMS. No patients with MS had a primary progressive disease course. Only one MS patient had an ADEM as first presentation. After dividing the MS patients into two groups, aged over or below 11 years, the sex ratio showed a trend towards significance (p < 0.001)  Table . The calAt last follow-up, 73/89 MS patients were on DMT for the duration of at least 1 year and in 28 patients second-line treatment was started , four with recurrent ON, two with multiphasic disseminated encephalomyelitis (MDEM) and one with ON followed by seizures   n = 2).The patients with multiphasic non-MS disease consisted of five patients who were diagnosed with ADEM-ON, five with NMOSD (AQP4 sy) Fig. . Of thesp < 0.001).Overall, physicians or parents reported at least one form of residual deficit at the last follow-up in 162/235 (69%) ADS patients, including 71/86 (83%) of the MS patients, 76/133 (57%) of the monophasic patients and 15/16 (94%) of the multiphasic non-MS patients. Residual neurological deficits were significantly more observed in MS patients compared to monophasic patients (p = 0.02). From the monophasic patients with a TM and residual deficits 11/14 had suffered from a longitudinal extended transverse myelitis. In MS and multiphasic non-MS patients, no significant difference was found in residual deficits between the presenting phenotypes.In the mono-ADS group, residual deficits were most often present in patients with TM and NMOSD (p < 0.05)  Fig. . Yet in Thirty-two percent of all included patients (78/243) underwent an NPA .At least one of the cognitive domains was affected in 65 of the tested patients (83%), respectively, in 29/32 (91%) monophasic ADS, 29/38 (76%) MS and 7/8 (88%) multiphasic non-MS. Three or more cognitive domains were affected in 18/32 (56%) monophasic patients, 22/38 (58%) MS patients and in 6/8 (75%) multiphasic non-MS patients. The three most commonly affected domains in monophasic patients were intelligence, attention and concentration and memory, in MS patients language, attention and concentration and memory and in multiphasic non-MS patients attention and concentration, memory and executive control functions. Median time till NPA from disease onset was 15 months (IQR 6–32). In monophasic patients the median time was 25 months (IQR 11–62), in MS patients 11 (IQR 6–24) and in multiphasic non-MS patients 10 (IQR 3–22).n = 124, MS: n = 76, multiphasic non-MS patients: n = 16). Negatively affected school performance was reported by 63/216 (29%) of the participants: 37/124 (30%) of the monophasic patients, 16/76 (21%) of the MS patients and 10/16 (63%) of the multiphasic non-MS patients. This included children to require academic accommodations, for instance extra assistance at school, extra time to complete examinations and change to special education.Data on school performance were available in 216/243 patients  of the monophasic patients had CI assessed through an NPA, 7/16 (44%) of the MS patients and 6/10 (60%) of the multiphasic non-MS patients.Furthermore, a total of 122/243 patients reported attention deficits in the standardized questionnaire.n = 3). These patients had a follow-up time of 24, 52 and 96 months and reached EDSS 5.5 at 6, 48 and 66 months, respectively. The first patient declined DMT. DMT was commenced in the other two patients, and both were escalated to second-line treatment  because of high MS disease activity.Three of the MS patients had an EDSS of 5.5 or above at the last moment of follow-up. All three patients received acute treatment at presentation. They all presented with a polyfocal CIS at the first event, including brainstem as well as spinal involvement . Remarkably 78% of these patients were tested seropositive for MOG-ab, in line with previous findings that MOG-ab positivity pleads against MS diagnosis and that these patients tend to have a relapsing disease course , 28.MOG-ab and AQP4-ab seropositivity may be underestimated in this cohort, as the CBAs for both antibodies were developed and validated after the start of our prospective study. Serum was not retrospectively available of every patient who was included before the CBAs were implemented in routine diagnostics.Over a median follow-up time of 61 months, only three MS patients reached an EDSS of 5.5 or above. However, residual neurological deficits are common in patients with MS (83%), in line with previous studies –16. CognOur data further feed the impression that one single hit of ADEM can leave considerable intracerebral damage and may be considered less benign than previously thought –34. A reThe high number of children with long-term residual deficits in the total group is concerning in relationship with school performances and psychomotor development, especially taking into account the cumulative nature of acquired disabilities after a longer disease duration in chronic demyelinating syndromes , 36. FutThere are some limitations to this study. Despite our quite unique and extensive national paediatric MS network with full geographical coverage, it is still possible that we have missed a few cases and thus have an underestimation of our incidence figures. Adolescents with CIS may have been assessed and followed up by an adult neurologist and, therefore, have not been referred to take part in our study.In addition, the negatively affected school performance in ADS patients may be correlated with other problems than CI. Fatigue, mood disorders, anxiety and negative coping strategies could correlate with a negative school performance in these patients, and may interact with cognitive impairment as well –16.In conclusion, the reported incidence of ADS and MS in the Netherlands has increased during the previous years. Across all ADS subtypes the observed residual neurological deficits are considerable. Long-term follow-up studies of ADS patients will be needed to provide more insight into the risks involved and to identify possibilities for timely intervention.


COMP) gene cause both pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED). Most mutations in COMP are located in the region encoding type 3 thrombospondin like domain (TSP3D). We report two Japanese boys with PSACH who had different novel in-frame deletions in TSP3D. The result recapitulates previous reports in that the in-frame deletions in TSP3D preferentially caused PSACH rather than MED.Mutations in the cartilage oligomeric matrix protein ( Mutations in the cartilage oligomeric matrix protein (COMP) gene give rise to a spectrum of disorders, severe PSACH, and mild multiple epiphyseal dysplasia (MED)4. COMP is a calcium-binding homopentameric protein that forms disulfide bonds between monomers5. The COMP monomer contains eight type-3 thrombospondin like domains 6, which is where approximately 85% of the COMP mutations were identified7. Each TSP3D has a consensus motif, where one or two calcium ion-binding consensus sequences (DXDXDGXXDXXD) reside. The number of binding calcium ions alters the conformation and function of the COMP protein8. Briggs et al.7 reported that missense mutations in T6, T7, and T8 and in-frame deletions in TSP3D preferentially cause PSACH rather than MED. We report two children with PSACH who each had a novel in-frame deletion.Pseudoachondroplasia  is a rare autosomal dominant chondrodysplasia characterized by specific clinical features, such as disproportionately short stature, brachydactyly, loose joints, ligamentous laxity, and early onset osteoarthritisThe proband visited our outpatient clinic at 6 years and 2 months of age. He was born to non-consanguineous Japanese parents at 40 weeks of gestation. The pregnancy and delivery were uncomplicated. His birth weight, length, and head circumference were 3570 g ( + 1.43 SD), 49.7 cm (+0.33 SD), and 34.8 cm (+0.96 SD), respectively. There was no family history of short stature or any skeletal diseases.Growth retardation was evident at 2 years of age. At 4 years, he was diagnosed with PSACH due to waddling gait, short limbs, and radiologic features, including oval-shaped platyspondyly, anterior breaking of vertebrae, small and irregular epiphyses, and irregular metaphyses Fig. .Fig. 1RaThe proband sought medical attention at 2 years and 9 months of age. He was born to non-consanguineous Japanese parents via vaginal delivery at 39 weeks and 1 day of gestation. The pregnancy and delivery were uncomplicated. His birth weight, length, and head circumference were 3274 g (+0.49 SD), 49.5 cm (+0.34 SD), and 34.0 cm (+0.59 SD), respectively. There was no family history of short stature or any skeletal diseases.He attained developmental milestones normally until 1 year of age. He presented initially with gait disturbance at 1 year and 9 months of age. He visited an orthopedic clinic, and metaphyseal irregularity and epiphyseal hypoplasia of the femur and tibia were noted. He was diagnosed as having PSACH based on his short stature (−2.18 SD), waddling gait, and radiologic findings, such as oval-shaped platyspondyly, anterior breaking of vertebrae, small and irregular epiphyses, and irregular metaphyses Fig. .COMP gene by Sanger sequencing. Primer sequences and PCR conditions are available upon request. This study was approved by the Ethics Committee of Keio University School of Medicine or Ethics Committee of Tokyo Metropolitan Children’s Medical Center. We obtained written informed consent for molecular studies from the parents. We assessed the pathogenicity of genomic variations of COMP using University of California Santa Cruz (UCSC) Genome Browser for evolutionary conservation, Human Gene Mutation Database (HGMD) for previous descriptions, and the Exome Aggregation Consortium (ExAC) database for allele frequency.Genomic DNA was extracted from peripheral white blood cells. We examined all coding exons and flanking introns of the We identified a heterozygous in-frame deletion in each case: a 12-base deletion in exon 13 (NM_000095(COMP_v001):c.1426_1437del [p.Gly476_Asp479del]) for case 1 Fig.  and a 9-COMP gene in PSACH children. Unfortunately, we were not able to ascertain that these deletions were de novo. However, it is very likely that both deletions are pathogenic because the deleted segments spanned amino acid residues that are conserved among different species and are compatible with the consensus motif in TSP3D.We reported two novel in-frame deletions in the 8. Likewise, it is tempting to assume that the removal of two amino acids in the calcium ion-binding consensus sequence due to the deletion in case 1 conspicuously impaired calcium ion affinity. The deleted amino acids in case 2 include cysteine 312, which is known to form a disulfide bond with cysteine 2929; thus, the mutant protein creates a severe conformational change.The deleted amino acids in case 1 reside in T7, which is a common deletion for PSACH (p.Asp469del). The common deletion removes a single amino acid in the calcium ion-binding consensus sequence. The biological consequence must be low affinity for calcium ion binding because the conformation of the COMP protein depends on the number of bound calcium ionsCOMP gene and confirmed the previous notion that in-frame deletion preferentially causes PSACH rather than MED.This study reported two novel pathogenic in-frame deletions in the The relevant data from this Data Report are hosted at the Human Genome Variation Database at 10.6084/m9.figshare.hgv.2318 and 10.6084/m9.figshare.hgv.2321.