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Introduction: The impact of residual internal fixation devices on subsequent procedures about the hip has not been clearly well defined. The objective of the current study is to evaluate the outcome of hip arthroplasty after hardware retrieval as a one-stage replacement, to analyze possible differences related to the type of removed implant, and to assess the impact of unexpected intraoperative cultures during implant retrieval., Materials and Methods: We present a retrospective study including all those cases undergoing hip arthroplasty with concomitant hardware removal (cannulated screws, intramedullary nail, or dynamic hip screw) from 2005 to 2018. We evaluated demographics, intraoperative cultures, early infection rate, and other complications., Results: A total of 55 cases were included in the study. The median time between the implant surgery and the hip arthroplasty was 113 days. The removed devices included 6 cannulated screws, 34 intramedullary nails, and 15 dynamic hip screws. Up to 74.5% of the failed osteosynthesis belonged to intertrochanteric femoral fractures. Dislocation rate was 9.1% (1.8% requiring revision surgery), 25.5% of the cases needed further new surgeries after the hip arthroplasty, and 49.1% died during the follow-up period. Any-cause revision surgery and mortality rates were significantly increased after intramedullary nail removal. Intraoperative cultures were performed in 46 cases, and in 9 (16.4%), there was bacterial contamination: 6 cases (10.9%) presented one single positive culture and 3 (5.5%) presented ≥ 2 positive cultures for the same microorganism. A total of five cases (9.1%) presented early prosthetic joint infection that required debridement. None of these five cases had presented positive cultures at the implant removal., Conclusion: According to our results, hip arthroplasty with concomitant hardware removal is related to a high 5-year mortality rate, mainly when intramedullary nail is retrieved. Whereas a high risk of early prosthetic joint infection is associated, it seems not to be related to the elevated presence of unexpected positive cultures.

CHEK2 variants are associated with intermediate breast cancer risk, among other cancers. We aimed to comprehensively describe CHEK2 variants in a Spanish hereditary cancer (HC) cohort and adjust the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) guidelines for their classification. First, three CHEK2 frequent variants were screened in a retrospective Hereditary Breast and Ovarian Cancer cohort of 516 patients. After, the whole CHEK2 coding region was analyzed by next-generation sequencing in 1848 prospective patients with HC suspicion. We refined ACMG-AMP criteria and applied different combined rules to classify CHEK2 variants and define risk alleles. We identified 10 CHEK2 null variants, 6 missense variants with discordant interpretation in ClinVar database, and 35 additional variants of unknown significance. Twelve variants were classified as (likely)-pathogenic; two can also be considered "established risk-alleles" and one as "likely risk-allele." The prevalence of (likely)-pathogenic variants in the HC cohort was 0.8% (1.3% in breast cancer patients and 1.0% in hereditary nonpolyposis colorectal cancer patients). Here, we provide ACMG adjustment guidelines to classify CHEK2 variants. We hope that this study would be useful for variant classification of other genes with low effect variants., (© 2020 Wiley Periodicals LLC.)

RNA analyses are a potent tool to identify spliceogenic effects of DNA variants, although they are time-consuming and cannot always be performed. We present splicing assays of 20 variants that represent a variety of mutation types in 10 hereditary cancer genes and attempt to incorporate these results into American College of Medical Genetics and Genomics (ACMG) classification guidelines. Sixteen single-nucleotide variants, 3 exon duplications, and 1 single-exon deletion were selected and prioritized by in silico algorithms. RNA was extracted from short-term lymphocyte cultures to perform RT-PCR and Sanger sequencing, and allele-specific expression was assessed whenever possible. Aberrant transcripts were detected in 14 variants (70%). Variant interpretation was difficult, especially comparing old classification standards to generic ACMG guidelines and a proposal was devised to weigh functional analyses at RNA level. According to the ACMG guidelines, only 12 variants were reclassified as pathogenic/likely pathogenic because the other two variants did not gather enough evidence. This study highlights the importance of RNA studies to improve variant classification. However, it also indicates the challenge of incorporating these results into generic ACMG guidelines and the need to refine these criteria gene specifically. Nevertheless, 60% of variants were reclassified, thus improving genetic counseling and surveillance for carriers of these variants., (Copyright © 2020 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: The BRCA1 c.3331_3334delCAAG founder mutation has been reported in hereditary breast and ovarian cancer families from multiple Hispanic groups. We aimed to evaluate BRCA1 c.3331_3334delCAAG haplotype diversity in cases of European, African, and Latin American ancestry., Methods: BC mutation carrier cases from Colombia (n = 32), Spain (n = 13), Portugal (n = 2), Chile (n = 10), Africa (n = 1), and Brazil (n = 2) were genotyped with the genome-wide single nucleotide polymorphism (SNP) arrays to evaluate haplotype diversity around BRCA1 c.3331_3334delCAAG. Additional Portuguese (n = 13) and Brazilian (n = 18) BC mutation carriers were genotyped for 15 informative SNPs surrounding BRCA1. Data were phased using SHAPEIT2, and identical by descent regions were determined using BEAGLE and GERMLINE. DMLE+ was used to date the mutation in Colombia and Iberia., Results: The haplotype reconstruction revealed a shared 264.4-kb region among carriers from all six countries. The estimated mutation age was ~ 100 generations in Iberia and that it was introduced to South America early during the European colonization period., Conclusions: Our results suggest that this mutation originated in Iberia and later introduced to Colombia and South America at the time of Spanish colonization during the early 1500s. We also found that the Colombian mutation carriers had higher European ancestry, at the BRCA1 gene harboring chromosome 17, than controls, which further supported the European origin of the mutation. Understanding founder mutations in diverse populations has implications in implementing cost-effective, ancestry-informed screening.

Acrylamide, a compound identified as a probable carcinogen, is generated during the sterilization phase employed during the processing of Californian-style green ripe olives. It is possible to reduce the content of this toxic compound by applying different strategies during the processing of green ripe olives. The influence of different processing conditions on acrylamide content was studied in three olives varieties ("Manzanilla de Sevilla", "Hojiblanca", and "Manzanilla Cacereña"). Olives harvested during the yellow-green stage presented higher acrylamide concentrations than green olives. A significant reduction in acrylamide content was observed when olives were washed with water at 25 °C for 45 min (25% reduction) and for 2 h (45% reduction) prior to lye treatment. Stone olives had 21-26% higher acrylamide levels than pitted olives and 42-50% higher levels than sliced olives in the three studied varieties. When calcium chloride (CaCl 2 ) was added to the brine and brine sodium chloride (NaCl) increased from 2% to 4%, olives presented higher concentrations of this contaminant. The addition of additives did not affect acrylamide levels when olives were canned without brine. Results from this study are very useful for the table olive industry to identify critical points in the production of Californian-style green ripe olives, thus, helping to control acrylamide formation in this foodstuff.

Near infrared spectroscopy (NIRS) and mid-infrared spectroscopy (MIRS) in combination with chemometric analysis were applied to discriminate the geographical origin of grapevine leaves belonging to the variety "Touriga Nacional" during different vegetative stages. Leaves were collected from plants of two different wine regions in Portugal (Dão and Douro) over the grapes maturation period. A sampling plan was designed in order to obtain the most variability within the vineyards taking into account variables such as: solar exposition, land inclination, altitude and soil properties, essentially. Principal component analysis (PCA) was used to extract relevant information from the spectral data and presented visible cluster trends. Results, both with NIRS and MIRS, demonstrate that it is possible to discriminate between the two geographical origins with an outstanding accuracy. Spectral patterns of grapevine leaves show significant differences during grape maturation period, with a special emphasis between the months of June and September. Additionally, the quantification of total chlorophyll and total polyphenol content from leaves spectra was attempted by both techniques. For this purpose, partial least squares (PLS) regression was employed. PLS models based on NIRS and MIRS, both demonstrate a statistically significant correlation for the total chlorophyll (R 2 P  = 0.92 and R 2 P  = 0.76, respectively). However, the PLS model for the total polyphenols, may only be considered as a screening method, because significant prediction errors, independently of resourcing on NIRS, MIRS or both techniques simultaneously, were obtained.

Phenylalanine (Phe) is a direct precursor of tyrosine and several neurotransmitters. The accumulation of Phe in the brain generates serious and not recoverable pathologies in children. Early detection in newborns is fundamental to apply the appropriate therapy and avoid irreversible health problems. Although fluorescence is a sensitive and selective technique for the determination of amino acids, the fluorescent analysis of Phe is limited since it exhibits a very low fluorescence quantum yield; however, the fluorescence of Phe increases drastically under UV irradiation when a peroxide medium is used. The aim of this research was to analyze the effect of the UV-radiation on Phe aqueous-peroxide solutions and to study the influence of the chemical environment on the photoinducted fluorescence process. The nature and characteristics of the fluorescent photoproducts generated under off-line UV irradiation in hydrogen peroxide medium were achieved by high performance liquid chromatography (HPLC) using a spectrophotometer detector (DAD) coupled in series with a mass spectrometer (MS) or with a fast scan spectrofluorimetric detector (FSFD). Environmental characteristics such as pH, initial concentration of Phe, hydrogen peroxide amount and irradiation time were studied in order to establish their influence on the formation of each one of the photoproducts. As the formation of several highly fluorescent photoproducts has been confirmed, the possibility of designing a chromatographic system with a post-column on-line photoreactor is open. The measure of the total fluorescence signal generated from Phe at the optimized irradiation time, could be used for the determination, with high sensitivity, of the initial amount of Phe in aqueous media, such as human serum or environmental samples. These aspects are being studied at present. .

The multifactorial likelihood analysis method has demonstrated utility for quantitative assessment of variant pathogenicity for multiple cancer syndrome genes. Independent data types currently incorporated in the model for assessing BRCA1 and BRCA2 variants include clinically calibrated prior probability of pathogenicity based on variant location and bioinformatic prediction of variant effect, co-segregation, family cancer history profile, co-occurrence with a pathogenic variant in the same gene, breast tumor pathology, and case-control information. Research and clinical data for multifactorial likelihood analysis were collated for 1,395 BRCA1/2 predominantly intronic and missense variants, enabling classification based on posterior probability of pathogenicity for 734 variants: 447 variants were classified as (likely) benign, and 94 as (likely) pathogenic; and 248 classifications were new or considerably altered relative to ClinVar submissions. Classifications were compared with information not yet included in the likelihood model, and evidence strengths aligned to those recommended for ACMG/AMP classification codes. Altered mRNA splicing or function relative to known nonpathogenic variant controls were moderately to strongly predictive of variant pathogenicity. Variant absence in population datasets provided supporting evidence for variant pathogenicity. These findings have direct relevance for BRCA1 and BRCA2 variant evaluation, and justify the need for gene-specific calibration of evidence types used for variant classification., (© 2019 Wiley Periodicals, Inc.)

Importance: Genetic testing of hereditary cancer using comprehensive gene panels can identify patients with more than one pathogenic mutation in high and/or moderate-risk-associated cancer genes. This phenomenon is known as multilocus inherited neoplasia alleles syndrome (MINAS), which has been potentially linked to more severe clinical manifestations., Objective: To determine the prevalence and clinical features of MINAS in a large cohort of adult patients with hereditary cancer homogeneously tested with the same gene panel., Patients and Methods: A cohort of 1023 unrelated patients with suspicion of hereditary cancer was screened using a validated panel including up to 135 genes associated with hereditary cancer and phakomatoses., Results: Thirteen (1.37%) patients harbouring two pathogenic mutations in dominant cancer-predisposing genes were identified, representing 5.7% (13/226) of patients with pathogenic mutations. Most (10/13) of these cases presented clinical manifestations associated with only one of the mutations identified. One case showed mutations in MEN1 and MLH1 and developed tumours associated with both cancer syndromes. Interestingly, three of the double mutants had a young age of onset or severe breast cancer phenotype and carried mutations in moderate to low-risk DNA damage repair-associated genes; two of them presented biallelic inactivation of CHEK2 . We included these two patients for the sake of their clinical interest although we are aware that they do not exactly fulfil the definition of MINAS since both mutations are in the same gene., Conclusions and Relevance: Genetic analysis of a broad cancer gene panel identified the largest series of patients with MINAS described in a single study. Overall, our data do not support the existence of more severe manifestations in double mutants at the time of diagnosis although they do confirm previous evidence of severe phenotype in biallelic CHEK2 and other DNA repair cancer-predisposing genes., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: The aim of the present study was to evaluate the association between prosthetic joint infection (PJI) after joint arthroplasty and the length of red blood cell (RBC) storage, timing of RBC transfusion, and the number of RBC units transfused. Study design and Methods: All patients who underwent a primary or revision joint artrhoplasty between January 2000 and December 2012 were retrospectively reviewed. For this study, only patients who received RBC transfusions during the day of the surgery (early transfusion group) or within the first 4 days after surgery (late transfusion group) were included. Results: A total of 9906 patients were reviewed. In the early transfusion group (n=1153, 11.6%), patients receiving 1 or 2 RBC units (3.5% vs 6.3%, P=0.041), 3 or 4 RBC (1.3% vs 13.3%, P=0.004) or ≥5 RBC units (5.0% vs 37.5%, P=0.026) had a higher PJI rate only when >50% of RBC units transfused had been stored >14 days. In the late transfusion group (n=920, 9.3%) these differences were not significant. Early transfusion of RBCs stored >14 days was an independent variable associated with an increased risk of PJI (OR:2.50, 95%CI:1.44-4.33) Conclusion: Transfusion of RBC within the first 6h after joint arthroplasty was an independent variable associated with PJI risk when RBC units are stored >14 days. The rate of PJI increased with the number of old RBC units transfused within this critical period., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© Ivyspring International Publisher.)

Background: Early joint decompression associated to antibiotic therapy is the most important procedure to reduce joint damage in septic knee arthritis in children. Several joint decompression methods have been described such as arthrotomy with open debriding, arthroscopic drainage or needle joint aspiration. The aim of the present study was to determinate which patients with acute septic knee arthritis could be safely treated with needle joint aspiration., Methods: Patients with an acute knee arthritis diagnosed between September 2003 and December 2013 in our children's tertiary hospital were retrospective review. All cases were initially treated with needle joint aspiration. Primary end-point was failure of joint aspiration., Results: A total of 74 patients were included in the study. Forty-two (56.8%) were male and median age was 1.49 years. Mean delay between onset of symptoms and diagnosis was 3.6 days and in 25 (33.8%) cases patients needed more than 1 visit to the emergency room. Median C-reactive protein (CRP) value was 36.3 mg/L and was >20 mg/L in 59 (79.7%) cases. A total of 11 (14.9%) patients showed failure of the joint aspiration treatment between 3 and 21 days after initial joint aspiration. The stepwise forward logistic regression model only identified as independent predictor of joint aspiration failure an age older than 3 years old (odds ratio, 5.64; 95% confidence interval, 1.38-29.61; P=0.018). Joint aspiration did not fail in any patient younger than 12 months and neither in any patient younger than 3 years old with CRP value <20 mg/L. Otherwise, treatment failed in 38% of patients older than 3 years and in 16% of patients between 1 and 3 years with a CRP>20 mg/L., Conclusions: Septic knee arthritis treated with needle joint aspiration succeed in all patients younger than 1 year and in all patients between 1 and 3 years with a CRP<20 mg/L. Alternative treatment such as arthroscopy debridement should be early considered in patients older than 3 years and patients between 1 and 3 years with CRP>20 mg/L., Level of Evidence: Level III.

Phenanthrene is the most released polycyclic aromatic hydrocarbon into the environment by anthropogenic action. Because of the absorption and biotransformation pathways, this compound is metabolized and the most abundant metabolites are hydroxylated derivatives, such as 1-, 2-, 3-, 4- and 9-hydroxyphenanthrene, which are excreted through biological fluids, included mammals milk. For the resolution and quantitation of co-eluted analytes, elution time-emission fluorescence matrices were analysed with different second-order calibration algorithms: n-way and unfolded partial least squares, both coupled with residual bilinearization (N-PLS/RBL and U-PLS/RBL), and multivariate curve resolution-alternative least squares (MCR-ALS). Once optimized the chromatographic parameters, in isocratic mode, the elution time was of 5.5 min. The second-order data were obtained exciting at 250 nm, with an emission range from 330 to 430 nm, each 1 nm, and elution time from 0 to 5.5 min each 5.4 s. The ranges for the second-order multivariate methods in validation samples were from 1.0 to 9.0 ng mL -1 for 1-, 2-, 3- and 4-hydroxyphenanthrene, and from 5.0 to 45.0 ng mL -1 for 9-hydroxyphenanthrene. Root mean square errors of prediction between 0.45 and 1.82 ng mL -1 (relative errors of prediction 7-22%) were obtained. The optimized procedures were applied in the analysis of human breast milk and in whole and semi-skimmed commercial cow milk. N-PLS/RBL and U-PLS/RBL algorithms show satisfactory results for the five metabolites with recoveries ranging between 82% and 115%., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Debridement, antibiotics, and implant retention (DAIR) is a widely used treatment modality for early acute prosthetic joint infection (PJI). A preoperative risk score was previously designed for predicting DAIR failure, consisting of chronic renal failure (K), liver cirrhosis (L), index surgery (I), cemented prosthesis (C), and C-reactive protein >115 mg/L (KLIC). The aim of this study was to validate the KLIC score in an external cohort., Methods: We retrospectively evaluated patients with early acute PJI treated with DAIR between 2006 and 2016 in 3 Dutch hospitals. Early acute PJI was defined as <21 days of symptoms and DAIR performed within 90 days after index surgery. Failure was defined as the need for (1) second DAIR, (2) implant removal, (3) suppressive antimicrobial treatment, or (4) infection-related death within 60 days after debridement., Results: A total of 386 patients were included. Failure occurred in 148 patients (38.3%). Patients with KLIC scores of ≤2, 2.5-3.5, 4-5, 5.5-6.5, and ≥7 had failure rates of 27.9%, 37.1%, 49.3%, 54.5%, and 85.7%, respectively (P < .001). The receiver-operating characteristic curve showed an area under the curve of 0.64 (95% confidence interval 0.59-0.69). A KLIC score higher than 6 points showed a specificity of 97.9%., Conclusion: The KLIC score is a relatively good preoperative risk score for DAIR failure in patients with early acute PJI and appears to be most useful in clinical practice for patients with low or high KLIC scores., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Understanding the mechanisms of cancer therapeutic resistance is fundamental to improving cancer care. There is clear benefit from chemotherapy in different breast cancer settings; however, knowledge of the mutations and genes that mediate resistance is incomplete. In this study, by modeling chemoresistance in patient-derived xenografts (PDXs), we show that adaptation to therapy is genetically complex and identify that loss of transcription factor 4 (TCF4; also known as ITF2) is associated with this process. A triple-negative BRCA1 -mutated PDX was used to study the genetics of chemoresistance. The PDX was treated in parallel with four chemotherapies for five iterative cycles. Exome sequencing identified few genes with de novo or enriched mutations in common among the different therapies, whereas many common depleted mutations/genes were observed. Analysis of somatic mutations from The Cancer Genome Atlas (TCGA) supported the prognostic relevance of the identified genes. A mutation in TCF4 was found de novo in all treatments, and analysis of drug sensitivity profiles across cancer cell lines supported the link to chemoresistance. Loss of TCF4 conferred chemoresistance in breast cancer cell models, possibly by altering cell cycle regulation. Targeted sequencing in chemoresistant tumors identified an intronic variant of TCF4 that may represent an expression quantitative trait locus associated with relapse outcome in TCGA. Immunohistochemical studies suggest a common loss of nuclear TCF4 expression post-chemotherapy. Together, these results from tumor xenograft modeling depict a link between altered TCF4 expression and breast cancer chemoresistance., Competing Interests: Competing interestsA. Villanueva and M.A.P. are recipients of unrestricted research grants from Roche Pharma and Astellas Pharma. A. Vidal and A. Villanueva are co-founders of Xenopat. No potential conflicts of interest were disclosed by the other authors., (© 2018. Published by The Company of Biologists Ltd.)

The original version of this article unfortunately contained a mistake. The presentation of the article title and subtitle was incorrect.

According to kin selection theory, indirect reproductive advantages may induce individuals to care for others with whom they share genes by common descent, and the amount of care, including self-sacrifice, will increase with the proportion of genes shared. Twins represent a natural situation in which this hypothesis can be tested. Twin pairs experience the same early environment because they were born and raised at the same time and in the same family but their genetic relatedness differs depending on zygosity. We compared the degree of willingness to fight and sacrifice for the co-twin among monozygotic (MZ) and dizygotic (DZ) pairs in a sample of 1443 same-sex and opposite-sex twins. We also analyzed the effect of the subject's gender and that of the co-twin on those altruistic behaviors. Results partly supported the postulated explanation. MZ twins (who share nearly their entire genome) were significantly more likely than DZ twins (who on average share half of their segregating genes) to self-sacrifice for their co-twins, but zygosity did not affect willingness to fight for him/her. The genders of the subject and of the co-twin, not genetic relatedness, were the best predictors of aggressive altruistic intentions.

Objectives: The combination of a fluoroquinolone with rifampin is one of the cornerstones in the treatment of prosthetic joint infections (PJI) caused by staphylococci. Moxifloxacin is highly active against methicillin-susceptible Staphylococcus aureus (MSSA) and, therefore, is an attractive agent to use. However, several studies reported a lowering in serum moxifloxacin levels when combined with rifampin. The clinical relevance remains unclear. We determined the outcome of patients with early acute PJI caused by MSSA treated with either moxifloxacin/rifampin or levofloxacin/rifampin., Methods: Medical files of patients treated with moxifloxacin/rifampin (University Medical Centre Groningen) or levofloxacin/rifampin (Hospital Clinic Barcelona) were retrospectively reviewed (2005-2015). Treatment failure was defined as the need for revision surgery and/or suppressive therapy, death by infection or a relapse of infection during follow-up., Results: Differences in baseline characteristics between the two cohorts were observed, but prognostic parameters for failure, as defined by the KLIC-score (Kidney failure, Liver cirrhosis, Index surgery, C-reactive protein and Cemented prosthesis), were similar in the two groups (2.9 [1.5 SD] for the moxifloxacin group vs. 2.2 [1.2 SD] for the levofloxacin group [P = 0.16]). With a mean follow-up of 50 months (36 SD) in the moxifloxacin group, and 67 months (50 SD) in the levofloxacin group (P = 0.36), treatment was successful in 89% vs. 87.5%, respectively (P = 0.89). None of the failures in the moxifloxacin group were due to rifampin- or moxifloxacin-resistant S. aureus strains., Conclusion: Our data indicate that moxifloxacin combined with rifampin is as clinically effective as levofloxacin/rifampin for early acute PJI caused by MSSA., (Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

Background: A significant amount of patients undergoing revision surgery of a prosthetic joint turn out to have an infection. Withholding preoperative antibiotic prophylaxis in these patients to optimize culture yield during revision surgery remains a matter of debate. The aim of our study was to determine (1) the rate of positive intraoperative cultures with or without preoperative antibiotic prophylaxis and (2) the incidence of a prosthetic joint infection (PJI) during the follow-up in the 2 groups., Methods: Medical files of patients in whom preoperative antibiotic prophylaxis was withheld until culture samples were taken (2007-2010, n = 284) and in whom antibiotic prophylaxis was given during the induction of anesthesia (2010-2013, n = 141) were retrospectively reviewed., Results: The percentage of ≥1 positive cultures was the same in the group without (26%) and with preoperative prophylaxis (27%; P value, .7). PJI was diagnosed during revision surgery according to the Musculoskeletal Infection Society criteria in 6.7% patients not receiving preoperative prophylaxis and in 7.0% receiving it (P value, .79). We found no important differences in the type of microorganisms that were isolated in both groups. During a 3-month follow-up, an early PJI developed in patients undergoing total revision surgery in 6.4% of the nonpreoperative prophylaxis group vs 1.6% in the preoperative prophylaxis group (P value, .1)., Conclusion: Preoperative antibiotic prophylaxis does not reduce culture yield in patients undergoing knee revision surgery. Our data show a trend toward a higher PJI rate in the postoperative period of total revision surgery when preoperative prophylaxis is withheld., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Mastocytosis comprises a heterogeneous group of disorders characterized by mast cell accumulation and proliferation in distinct organs. Kounis syndrome is defined as the concurrence of acute coronary syndromes with mast cell activation in a setting of allergic or hypersensitivity reactions. This is the first reported case of an intraoperative Kounis syndrome as the onset of an indolent systemic mastocytosis probably triggered by succinylated gelatin infusion during general anesthesia. The presentation of this case is intended to contribute to the knowledge of mastocytosis and Kounis syndrome at the time of diagnostic workup during intraoperative anaphylaxis or myocardial ischemia.

We wanted to implement an NGS strategy to globally analyze hereditary cancer with diagnostic quality while retaining the same degree of understanding and control we had in pre-NGS strategies. To do this, we developed the I2HCP panel, a custom bait library covering 122 hereditary cancer genes. We improved bait design, tested different NGS platforms and created a clinically driven custom data analysis pipeline. The I2HCP panel was developed using a training set of hereditary colorectal cancer, hereditary breast and ovarian cancer and neurofibromatosis patients and reached an accuracy, analytical sensitivity and specificity greater than 99%, which was maintained in a validation set. I2HCP changed our diagnostic approach, involving clinicians and a genetic diagnostics team from panel design to reporting. The new strategy improved diagnostic sensitivity, solved uncertain clinical diagnoses and identified mutations in new genes. We assessed the genetic variation in the complete set of hereditary cancer genes, revealing a complex variation landscape that coexists with the disease-causing mutation. We developed, validated and implemented a custom NGS-based strategy for hereditary cancer diagnostics that improved our previous workflows. Additionally, the existence of a rich genetic variation in hereditary cancer genes favors the use of this panel to investigate their role in cancer risk.

Next generation sequencing panels have been developed for hereditary cancer, although there is some debate about their cost-effectiveness compared to exome sequencing. The performance of two panels is compared to exome sequencing. Twenty-four patients were selected: ten with identified mutations (control set) and fourteen suspicious of hereditary cancer but with no mutation (discovery set). TruSight Cancer (94 genes) and a custom panel (122 genes) were assessed alongside exome sequencing. Eighty-three genes were targeted by the two panels and exome sequencing. More than 99% of bases had a read depth of over 30x in the panels, whereas exome sequencing covered 94%. Variant calling with standard settings identified the 10 mutations in the control set, with the exception of MSH6 c.255dupC using TruSight Cancer. In the discovery set, 240 unique non-silent coding and canonic splice-site variants were identified in the panel genes, 7 of them putatively pathogenic (in ATM, BARD1, CHEK2, ERCC3, FANCL, FANCM, MSH2). The three approaches identified a similar number of variants in the shared genes. Exomes were more expensive than panels but provided additional data. In terms of cost and depth, panels are a suitable option for genetic diagnostics, although exomes also identify variants in non-targeted genes.

Background: The aim of our study was the early detection and treatment of patients with unknown alterations of the hydrocarbon metabolism subject to total knee arthroplasty in order to reduce the incidence of postoperative complications., Methods: Patients were classified as non-diabetic patients (group 1), diabetic patients (group 2) and patients with stress hyperglycaemia (group 3). The last two groups were recommended assessment by a primary care physician (PCP). After one year follow-up the groups were compared with respect to incidence of postoperative complications. The groups were also compared regarding the decrease or increase of HbA1c levels with the incidence of complications., Results: Of the 228 patients, 116 (50%) were included in group 1, 40 (17.5%) in group 2 and 72 (31.6%) in group 3. Patients that consulted their PCP presented lower medical complication rates than those who did not (9.2% vs. 26.4%, P=0.020). Not being attended by a PCP was an independent predictive factor of medical complication (odds ratio (OR): 21.3; 95% confidence interval (95% CI): 4.6-98.5), surgical site infection (OR: 4.1; 95% CI: 1.1-15.0) and mechanical complication (OR: 5.0; 95% CI: 1.3-18.8). A decrease of HbA1c value was related to less medical systemic complications (7.3% vs. 24.2%, P=0.035)., Conclusions: Patients with hyperglycaemia during the postoperative total knee arthroplasty period, who are controlled by the PCP present lower incidence of complications. Decrease of HbA1c value during postoperative total knee arthroplasty period leads to a lower rate of medical complications., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Large acetabular defects remain a challenge in hip revision arthroplasty. Experience with Trabecular Titanium™ (TT) cups (Limacorporate S.p.a.) has not been widely reported. Therefore, we assessed the survivorship and clinical and radiological outcomes of patients receiving TT cups, with or without supplementary trabecular titanium hemispherical modules for acetabular reconstruction, in primary and revision total hip arthroplasty (THA)., Methods: Between January 2009 and July 2014, we performed 67 revisions and 5 primary THAs using TT cups in 69 patients. To achieve stability and/or restore the hip's centre of rotation, hemispherical modules were used in 17 cases based on preoperative templating and/or intraoperative findings. Mean follow-up was 30.5 months. Acetabular bone defects were classified according to the Paprosky classification. Survivorship, functional outcomes (Merle d'Aubigné) and radiological outcomes were analysed., Results: 8 patients underwent cup revision: 2 for loosening, 3 for infection, and 3 for hip dislocation. The remaining cases did not present radiological signs of loosening. None of the cases with Paprosky type I classifications needed revision, while 4 with type II and 4 with type III needed revision (p = 0.028). The respective mean values for pain, walking, and range of motion (Merle d'Aubigné scores ± standard deviation) were 3.6 ± 1.4, 3.7 ± 0.7, and 3.8 ± 0.6 preoperatively, and 5.7 ± 0.7, 5.3 ± 0.7, and 5.6 ± 0.7 at the latest follow-up (p&lt;0.001)., Conclusions: In the short term, results with TT cups appear to be encouraging, with satisfactory survival rates for both simple and complex cases.

Background: Surgical site infection (SSI) rate after surgery for hip fracture is about 4%. The aim of the present study was to review the efficacy of dual prophylaxis using teicoplanin plus cefuroxime and risk factors for SSI., Patients: Operations for hip fracture from 2012 to 2013 were retrospectively reviewed. Relevant information was gathered: Demographics, comorbidities, ASA score, laterality, type of fracture, type of surgery, d from admission to surgery, length of surgery, hemoglobin value at admission, urinary or respiratory infections, and the need for pre-operative, intra-operative, and post-operative red blood cell (RBC) transfusion. Prophylaxis consisted of cefuroxime and teicoplanin during the induction of anesthesia. U.S. Centers for Disease Control and Prevention (CDC) criteria for superficial and deep SSI were applied. Univariate and multivariable analysis were performed., Results: Six hundred fifty-seven patients were included in the study. Thirteen (2.0%) SSI were identified, six superficial (0.9%), and seven deep (1.1%). Staphylococcus aureus was isolated in two infections (one superficial and one deep). The SSI rate was 2.4% in intra-medullary nails (n = 334), 1.4% in prostheses (n = 211), and 1.8% in other synthesis (n = 112). Parameters independently associated with SSI were: Intra-operative RBC transfusion (OR: 11.6, p = 0.002), length of surgery >120 min (OR: 4.5, p = 0.02), and having a urinary infection (OR: 4.28, p = 0.02)., Conclusion: Dual prophylaxis including cefuroxime and teicoplanin was associated with a 2% rate of SSI. Staphylococcus aureus caused only two SSIs. Reducing SSI is of utmost importance for patients' quality of life and to avoid additional cost of surgical procedures. Therefore, more experience with dual prophylaxis is needed to confirm our results.

The anatomy of the superficial peroneal nerve (SPN) and, more precisely, of the distal branches of the SPN at the ankle has attracted interest owing to the possibility of injury when performing ankle arthroscopy. The anterolateral portal is one of the most commonly used portals in ankle arthroscopy, and the intermediate dorsal cutaneous nerve can easily be injured during portal placement. The purpose of the present study was to assess whether visual inspection and palpation of the cutaneous nerves at the ankle differed from examination with ultrasonography and whether the 2 examination techniques correlated with the anatomic location of the SPN, which was verified by cadaver dissection. First, visual examination and palpation was performed to identify the SPN, after which 12 cadaver legs from separate specimens were examined with ultrasonography to mark the course of the SPN. We then measured the distance between the nerve as identified with gross visualization/palpation and ultrasound examination, and compared these with the precise location determined by anatomic dissection. The use of ultrasonography to determine the course of the SPN was good or excellent in 11 of the 12 legs (91.7%) studied. In contrast, gross visualization/palpation was good or excellent in 4 legs (33.3%). Excellent agreement was observed between the ultrasound markings and the anatomic dissection results. However, the visual examination poorly identified the course and the anatomic variations of the nerve branches evidenced in the anatomic dissection. From these findings in cadaver specimens, ultrasound identification of the SPN and its branches is likely preferable to gross visualization/palpation before placement of the anterolateral arthroscopic portal to the ankle., (Copyright © 2016 American College of Foot and Ankle Surgeons. Published by Elsevier Inc. All rights reserved.)

Purpose: Transfusion of packed red blood cells (RBCs) stored for &gt;14 days has been associated with a higher risk of infection but there is no data about the impact in revision hip arthroplasty., Methods: We retrospectively reviewed 280 patients who underwent revision hip arthroplasty from January 2002 to May 2012. Relevant risk factors and prosthetic joint infection (PJI) rate after 100 days after surgery were collected., Results: 10 patients out of 280 (3.6%) had a PJI within the first 100 days after revision surgery. The PJI rate in patients receiving an early transfusion (during surgery or within the first 12 hours) of packed RBCs with a length of storage &gt;14 days was higher (8.0%) than the rate in nontransfused patients (1.8%) or those receiving an early transfusion of packed RBCs with a length of storage ≤14 days (2.6%, p = 0.05). A Cox regression model identified transfusion of packed RBCs stored &gt;14 days as the only independent predictor of PJI (hazard ratio [HR] = 4.54; 95% confidence interval [CI], 1.13-18.15; p = 0.032)., Conclusions: The only independent predictor of PJI was early transfusion of packed RBCs stored &gt;14 days. Therefore, a potential way for reducing the PJI rate could be the selection of young packed RBCs.

Germline inactivating mutations in the BRCA1 and BRCA2 genes are responsible for hereditary breast and ovarian cancer syndrome (HBOCS). Genetic testing of these genes identifies a significant proportion of variants of uncertain significance (VUS). Elucidation of the clinical impact of these variants is an important challenge in genetic diagnostics and counseling. In this study, we assess the RNA effect of 28 BRCA1 and BRCA2 VUS identified in our set of HBOCS families with the aim of gaining insight into their clinical relevance. mRNA was extracted from VUS carriers and controls lymphocytes cultured for 5-6 days and treated with puromycin. RNA was reverse transcribed to perform transcriptional analysis for the study of splicing aberrations. In silico prediction tools were used to select those variants most likely to affect the RNA splicing process. Six out of the 28 variants analyzed showed an aberrant splicing pattern and could therefore be classified as probably pathogenic mutations. Reclassification of VUS improves the genetic counseling and clinical surveillance of carriers of these mutations and highlights the importance of RNA studies in routine diagnostic laboratories.

Background: The aim of this study was to compare the prosthetic joint infection (PJI) rate after knee revision arthroplasty in two consecutive periods with different antibiotic prophylaxis: short (one day) versus long (five days)., Methods: From January 2007 to September 2010 antibiotic prophylaxis consisted of 800 mg of teicoplanin and 2 g of ceftazidime intravenously and 1 g of ceftazidime two hours after the first dose. From October 2010, it was prolonged post-operatively using vancomycin and ceftazidime intravenously until the fifth day., Results: During the study period, 341 revision surgeries met the inclusion criteria. The PJI rate was lower in the long-prophylaxis group (2.2% versus 6.9%, p=0.049). Prolonged post-operative antibiotic treatment was the only variable associated independently with a lower rate of PJI (odds ratio [OR]: 0.27, 95% confidence interval [CI]: 0.07-0.99).

The aim of this study was to compare the prosthetic joint infection (PJI) rate after total joint arthroplasty in two consecutive periods of treatment with different antibiotic prophylaxes: cefuroxime versus cefuroxime plus teicoplanin. We retrospectively reviewed 1,896 patients who underwent total hip arthroplasty or total knee arthroplasty between March 2010 and February 2013. From March 2010 to August 2011, patients received 1.5 g of cefuroxime during induction of anesthesia and another 1.5 g 2 h later (the C group). From September 2011, 800 mg of teicoplanin was added to cefuroxime (the CT group). Throughout the period studied, there were no variations in pre- or postoperative protocols. Univariate and multivariate analyses were performed to evaluate independent predictors of PJI. There were 995 (55.7%) patients in the C group and 791 (44.3%) in the CT group. Patients in the CT group had a significantly lower PJI rate than patients in the C group (1.26% versus 3.51%, P=0.002). There were no infections due to Staphylococcus aureus in the CT group (0% versus 1.6% in the C group, P<0.001). A stepwise forward Cox regression model identified male sex (hazard ratio [HR], 3.85; 95% confidence interval [CI], 2.09 to 7.18), a body mass index of ≥35 kg/m2 (HR, 2.93; 95% CI, 1.37 to 6.27), the presence of lung disease (HR, 2.46; 95% CI, 1.17 to 5.15), and red blood cell transfusion (HR, 3.70; 95% CI, 1.89 to 7.23) to be independent variables associated with a higher risk of PJI. The addition of teicoplanin was associated with a lower risk of infection (HR, 0.35; 95% CI, 0.17 to 0.74). In conclusion, the addition of teicoplanin to cefuroxime during primary arthroplasty was associated with a significant reduction in the global PJI rate due to a reduction of infections caused by Gram-positive bacteria., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Purposes: The aim of the present study was to evaluate the importance of isolated microorganisms according to the Gram stain and the type of antibiotic received on the outcome of early prosthetic joint infection (PJI) treated with debridement, antibiotics and implant retention (DAIR)., Methods: From January 1999 to December 2009, all patients with an early PJI were prospectively registered in a database and they were retrospectively reviewed for this study., Results: During the study period, 160 patients met the inclusion criteria of the study. After a mean (SD) post-debridement follow-up of 5.2 (2.5) years, 117 patients (73.1%) were considered to be in remission and 43 (26.9%) were classified as failure. Variables associated with failure were liver cirrhosis (66.7% vs. 22.8%, p=0.001), diagnosis within the first 30 days from arthroplasty (30.4% vs. 8.0%, p=0.020), C-reactive protein (CRP) >12 mg/dl (46.7% vs. 21.2%, p=0.005), microorganism isolated in all deep samples (31.1% vs. 16.0%, p=0.047) and Gram-negative (GN) infection not treated with a fluoroquinolone (57.1% vs. 20.0%, P=0.004). Gram-positive (GP) infection not treated with rifampin was close to be statistically significantly associated with failure (34.4% vs. 19.2%, p=0.067). A multivariate analysis identified liver cirrhosis (OR: 12.4 CI95%: 3.1-49.7, p<0.001), CRP-value (OR: 1.06 CI95%: 1.0-1.11, p=0.049), and when a GN-infection was not treated with a fluoroquinolone (OR: 6.5, CI95%: 1.8-23.8, p=0.005) as independent predictors of failure., Conclusion: The remission rate of PJI treated with DAIR after 3 years of follow-up was 73%. The main predictors of failure were liver cirrhosis, the selected antibiotic most specially fluoroquinolones for GN and rifampin for GP infections, the C-reactive protein and the number of samples culture positive as a potential surrogate markers of bacterial density.

Introduction: Debridement and prosthesis retention, combined with a prolonged antibiotic regimen including rifampicin, is an accepted therapeutic approach when the duration of symptoms is less than 4 weeks and there are no radiological signs of loosening. The outcome of patients managed with this strategy has been previously assessed in several articles with success rates of 60-90%. This study aims to review the clinical experience with linezolid in 3 different hospitals from Spain and France in patients with prosthetic joint infection (PJI) managed with debridement, retention of the implant and treated with linezolid with or without rifampicin., Methods: Patients with an acute PJI who underwent open debridement with implant retention treated with linezolid for more than 7 days in 3 hospitals from Barcelona, Tours and Lille between 2005 and 2011 were retrospectively reviewed. Relevant information about demographics, co-morbidity, type of implant, surgical treatment, microorganism isolated, antimicrobial therapy, adverse events (AEs) and outcomes were recorded from patients., Results: A total of 39 patients were retrospectively reviewed. The mean age (SD) was 70.5 (8.8) years and 9 patients had diabetes mellitus (23%). There were 25 (64%) knee prostheses, 13 (33%) hips and 1 shoulder (3%). The median interquartile range (IQR) days from arthroplasty to infection diagnosis was 17 (19-48) and 33 (85%) cases were diagnosed within the first 60 days. The median (IQR) duration of antibiotic treatment was 70.5 (34-96) days and the median (IQR) number of days on linezolid treatment was 44.5 (30-81). AEs were observed in 15 patients (38%), with gastrointestinal complaints in 8 cases and anemia in 5 being the most frequent. After a median (IQR) follow-up of 2.5 (1.8-3.6) years, there were 11 failures (28%) (8 relapses and 3 new infections). The failure rate was higher in the rifampicin group (36% vs. 18%) mainly due to a higher relapse rate (27% vs. 12%) although differences were not statistically significant., Conclusion: Management of acute PJIs with debridement and retention of the implant linezolid, with or without rifampicin, is associated with a high remission rate and it is an alternative treatment for infections due to fluoroquinolone and/or rifampicin-resistant staphylococci.

The aim of our study was to determine the potential influence of blood transfusion and the length of storage of packed red blood cells (RBC) on prosthetic joint infection after primary knee arthroplasty. From November 2007 to November 2009, all variables potentially associated with deep infection were registered in 1331 consecutive patients who underwent total knee arthroplasty. Infection was diagnosed in 32 (2.4%) patients. After adjusting for important variables, blood transfusion with RBCs stored >14days was the strongest predictive factor for prosthetic joint infection within 90days after primary knee arthroplasty (OR: 5.9, 95% CI: 2.6-13.2, P < 0.001). Blood saving techniques are desirable to reduce perioperative blood transfusion., (Copyright © 2014 Elsevier Inc. All rights reserved.)

We aimed to analyze the efficacy and safety of high doses of daptomycin (10 mg/kg/d) plus rifampin (D10 + R) for prosthetic joint infection (PJI). This was an observational retrospective multicenter study (2010-2012) including all patients with acute PJI by fluoroquinolone-resistant staphylococci managed with implant retention and D10 + R. Twenty cases were included: 2 (10%) were withdrawn due to toxicity, leaving 18 cases for efficacy evaluation: 13 (72%) women, age 79 years (range 58-90). Clinical failure was observed in 9 (50%) patients: in 5 cases, staphylococci were recovered (28% of microbiological failures); no modification of daptomycin-MIC was observed. These 18 cases were compared with 44 matched historical cases: failure rate was similar, but whereas in the historical series, failure occurred fundamentally during therapy, in the present series, it was recorded after discontinuation of antibiotics. In summary, D10 + R may be the initial treatment of choice for PJI by fluoroquinolone-resistant staphylococci managed with implant retention., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Next-generation sequencing (NGS) has revolutionized genomic research and is set to have a major impact on genetic diagnostics thanks to the advent of benchtop sequencers and flexible kits for targeted libraries. Among the main hurdles in NGS are the difficulty of performing bioinformatic analysis of the huge volume of data generated and the high number of false positive calls that could be obtained, depending on the NGS technology and the analysis pipeline. Here, we present the development of a free and user-friendly Web data analysis tool that detects and filters sequence variants, provides coverage information, and allows the user to customize some basic parameters. The tool has been developed to provide accurate genetic analysis of targeted sequencing of common high-risk hereditary cancer genes using amplicon libraries run in a GS Junior System. The Web resource is linked to our own mutation database, to assist in the clinical classification of identified variants. We believe that this tool will greatly facilitate the use of the NGS approach in routine laboratories.

Objectives: To compare the specific characteristics, the outcome and the predictors of failure of prosthetic joint infections (PJI) due to methicillin-resistant (MRS) and methicillin- susceptible staphylococci (MSS) treated with open debridement and retention of the implant., Material and Methods: PJI due to MRS or MRS prospectively registered in a database from 1999 to 2009 were retrospectively reviewed., Results: During the study period, 96 patients met the inclusion criteria of the study. The mean follow-up period was 3.9 years and at least 2 years in all patients. The failure rate was 25%. The only variable significantly associated with failure in the global cohort was polymicrobial infection (59.3% vs. 40.7%, p=0.036). Thirty-four (35.4%) patients had an infection due to MRS and 62 (63.6%) due to MSS. Among MSS infections, 95.2% corresponded to primary arthroplasties while 29.4% of PJI due to MRS were after revision arthroplasties (p=0.001). CRP was significantly higher in PJI due to MSS (5.2 mg/dl vs 9.1 mg/dL, p=0.02).The failure rate (20% vs 27%, p=0.62) was very similar in MSS and MRS groups., Conclusion: PJI due to MRS were mainly coagulase-negative staphylococci, more frequent after revision arthroplasties, had a lower inflammatory response, and had a similar failure rate than MSS infections.

Next-generation sequencing (NGS) is changing genetic diagnosis due to its huge sequencing capacity and cost-effectiveness. The aim of this study was to develop an NGS-based workflow for routine diagnostics for hereditary breast and ovarian cancer syndrome (HBOCS), to improve genetic testing for BRCA1 and BRCA2. A NGS-based workflow was designed using BRCA MASTR kit amplicon libraries followed by GS Junior pyrosequencing. Data analysis combined Variant Identification Pipeline freely available software and ad hoc R scripts, including a cascade of filters to generate coverage and variant calling reports. A BRCA homopolymer assay was performed in parallel. A research scheme was designed in two parts. A Training Set of 28 DNA samples containing 23 unique pathogenic mutations and 213 other variants (33 unique) was used. The workflow was validated in a set of 14 samples from HBOCS families in parallel with the current diagnostic workflow (Validation Set). The NGS-based workflow developed permitted the identification of all pathogenic mutations and genetic variants, including those located in or close to homopolymers. The use of NGS for detecting copy-number alterations was also investigated. The workflow meets the sensitivity and specificity requirements for the genetic diagnosis of HBOCS and improves on the cost-effectiveness of current approaches.

Chronic systemic illnesses such as diabetes mellitus, chronic kidney disease (CKD), liver cirrhosis, neoplasia, etc. have been clearly associated with high rates of SWI. However, the exact mechanisms underlying these observations are still under investigation. Chronic kidney disease (CKD) is a growing problem in our society. Many of these patients will require an arthroplasty and it appears that the prosthetic infection risk for these types of patients is much higher than in the normal population. The risk of complications due to infection seems to be lower in patients with kidney transplants than in patients undergoing haemodialysis. Both prophylaxis and treatment of infection in patients with CKD should be carried out with a strict monitoring of potentially nephrotoxic antibiotics. The literature on the prognosis and risk of infection in patients with haematopoietic stem cell transplant is scarce and occasionally contradictory. The optimal time for the surgery should be determined by taking into account the immunological state of the patient and should be avoided, as much as possible, during the first year after the HSCT. Child's classification system is the most widely used method of stratifying the surgical risk for patients with cirrhosis; the infection appeared to be associated in a statistically significant way with advanced age and a Child B pre-operative classification. The prevention of prosthetic joint infections in HIV-infected patients should not be significantly different from the prevention for any other patient. Those patients that receive adequate antiretroviral treatment and periodic laboratory control show infection rates and periprosthetic complications that are similar to those for patients not affected by HIV. Therefore, the patient's level of immunodeficiency is the most important prognostic factor for prosthetic infection. The particular immunological condition of these patients can lead to infections due to particular microorganisms that immunocompetent patients do not have to deal with. Of all possibilities, because of their frequency and difficulty to treat, infections caused by methicillin-resistant S. aureus and fungus are highlighted.

Germline inactivating mutations in BRCA1 and BRCA2 genes are responsible for Hereditary Breast and Ovarian Cancer Syndrome (HBOCS). Genetic testing of these genes is available, although approximately 15% of tests identify variants of uncertain significance (VUS). Classification of these variants into pathogenic or non-pathogenic type is an important challenge in genetic diagnosis and counseling. The aim of the present study is to functionally assess a set of 7 missense VUS (Q1409L, S1473P, E1586G, R1589H, Y1703S, W1718L and G1770V) located in the C-terminal region of BRCA1 by combining in silico prediction tools and structural analysis with a transcription activation (TA) assay. The in silico prediction programs gave discrepant results making its interpretation difficult. Structural analysis of the three variants located in the BRCT domains (Y1703S, W1718L and G1770V) reveals significant alterations of BRCT structure. The TA assay shows that variants Y1703S, W1718L and G1770V dramatically compromise the transcriptional activity of BRCA1, while variants Q1409L, S1473P, E1586G and R1589H behave like wild-type BRCA1. In conclusion, our results suggest that variants Y1703S, W1718L and G1770V can be classified as likely pathogenic BRCA1 mutations.

A liquid chromatographic method with fluorimetric detection has been developed to determine the most abundant α-dicarbonyl compounds, generated as intermediates in the Maillard's reaction, previous derivatization to high fluorescent pteridinic derivatives. Hence, the biomarkers D-glucosone, 3-deoxyglucosone, glyoxal, methylglyoxal, diacetyl, 2,3-pentanedione, and phenylglyoxal were quantified using a gradient elution mode. The experimental conditions of the derivatization reaction and mobile phase composition were optimized. Linearity ranges (peak area versus α-dicarbonyl compound concentration) from 1.0 to 100.0 ng mL(-1) were obtained. Detection limits were comprised between 0.3 and 11.0 ng mL(-1). The high sensitivity of the method allows the determination of α-dicarbonyl compounds present in human urine, such as D-glucosone, 3-deoxyglucosone, glyoxal, and methylglyoxal, that are used as biomarkers, in order to investigate their roles in several diseases, with special emphasis in diabetes mellitus. With the aim of avoiding the interferences due to pteridinic compounds present in urine, a cleanup step with an ISOLUTE ENV+ cartridge was carried out. The concentrations of these urinary biomarkers have been reported as a normalized ratio to urinary creatinine, and determined in healthy and in diabetic volunteers, of different ages and sex. In all urine samples, standard addition and external calibration procedures were applied and compared., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Purposes: To evaluate the specific characteristics, outcome, and predictors of failure of prosthetic joint infections (PJI) due to S. aureus and coagulase-negative staphylococci (CNS) treated with open debridement and retention of the implant., Methods: PJI due to S. aureus or CNS prospectively registered in a database from 1999 to 2009 were retrospectively reviewed. During the study period, 106 patients met the inclusion criteria. The mean follow-up period was 3.8 years and for at least 2 years in all patients. The failure rate was 23.6% (25 out of 106). The only variable significantly associated with failure in the global cohort was polymicrobial infection (38.7% vs. 17.3%, p = 0.024). Fifty-seven (53.8%) patients had an infection due to S. aureus and 49 (46.2%) due to CNS. Among S. aureus infections, 95% corresponded to primary arthroplasties while 98% of PJIs due to CNS were after revision arthroplasties (p<0.001). C-reactive protein was significantly higher in PJI due to S. aureus (9.5 mg/dl vs. 4.9 mg/dl, p = 0.007). The rate of methicillin-resistance (8.8% vs. 59.2%, p<0.001) and fluoroquinolone-resistance (15.8% vs. 34.7%, p = 0.005) was significantly higher in CNS infections. The global failure rate was higher in S. aureus infections (28% vs. 18.3. p = 0.26). In S. aureus infections, patients diagnosed within the first 15 days after joint arthroplasty (p = 0.031) and with bacteremia (p = 0.046) had poor pro-gnosis. In CNS infections only the location of the prosthesis (knee 27.6% vs. hip 5%, p = 0.045) was associated with failure., Conclusions: PJIs due to S. aureus were mainly in primary arthroplasties; they had a higher inflammatory response; and the strains were more susceptible to fluoroquinolones and methicillin than CNS infections. S. aureus infections had a higher failure rate than CNS infections, however, the difference was not statistically significant. There were few factors associated with failure and they were different in S. aureus and CNS infections.

Mutations in BRCA1 and BRCA2 predispose carriers to early onset breast and ovarian cancer. A common problem in clinical genetic testing is interpretation of variants with unknown clinical significance. The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium was initiated to evaluate and implement strategies to characterize the clinical significance of BRCA1 and BRCA2 variants. As an initial project of the ENIGMA Splicing Working Group, we report splicing and multifactorial likelihood analysis of 25 BRCA1 and BRCA2 variants from seven different laboratories. Splicing analysis was performed by reverse transcriptase PCR or mini gene assay, and sequencing to identify aberrant transcripts. The findings were compared to bioinformatic predictions using four programs. The posterior probability of pathogenicity was estimated using multifactorial likelihood analysis, including co-occurrence with a deleterious mutation, segregation and/or report of family history. Abnormal splicing patterns expected to lead to a non-functional protein were observed for 7 variants (BRCA1 c.441+2T>A, c.4184&#95;4185+2del, c.4357+1G>A, c.4987-2A>G, c.5074G>C, BRCA2 c.316+5G>A, and c.8754+3G>C). Combined interpretation of splicing and multifactorial analysis classified an initiation codon variant (BRCA2 c.3G>A) as likely pathogenic, uncertain clinical significance for 7 variants, and indicated low clinical significance or unlikely pathogenicity for another 10 variants. Bioinformatic tools predicted disruption of consensus donor or acceptor sites with high sensitivity, but cryptic site usage was predicted with low specificity, supporting the value of RNA-based assays. The findings also provide further evidence that clinical RNA-based assays should be extended from analysis of invariant dinucleotides to routinely include all variants located within the donor and acceptor consensus splicing sites. Importantly, this study demonstrates the added value of collaboration between laboratories, and across disciplines, to collate and interpret information from clinical testing laboratories to consolidate patient management.

Comprehensive genetic testing of the breast cancer susceptibility genes BRCA1 and BRCA2 identified approximately 16% of variants of unknown significance (VUS), a significant proportion of which could affect the correct splicing of the genes. Our aim is to establish a workflow for classifying VUS in these complex genes, the first stage of which is splicing analysis. We used a combined approach consisting of five in silico splicing prediction programs and RT-PCR analysis for a set of 26 variants not previously studied at the mRNA level and six variants that had already been studied, four of which were used as positive controls as they were found to affect the splicing of these genes and the other two were used as negative controls. We identified a splicing defect in 8 of the 26 newly studied variants and ruled out splicing alteration in the remaining 18 variants. The results for the four positive and the two negative control variants were consistent with results presented in the literature. Our results strongly suggest that the combination of RNA analysis and in silico programs is an important step towards the classification of VUS. The results revealed a very high correlation between experimental data and in silico programs when using tools for predicting acceptor/donor sites but a lower correlation in the case of tools for identifying ESE elements.

Germline mutations in BRCA1 and BRCA2 are associated with increased risks of breast and ovarian cancer. A genome-wide association study (GWAS) identified six alleles associated with risk of ovarian cancer for women in the general population. We evaluated four of these loci as potential modifiers of ovarian cancer risk for BRCA1 and BRCA2 mutation carriers. Four single-nucleotide polymorphisms (SNPs), rs10088218 (at 8q24), rs2665390 (at 3q25), rs717852 (at 2q31), and rs9303542 (at 17q21), were genotyped in 12,599 BRCA1 and 7,132 BRCA2 carriers, including 2,678 ovarian cancer cases. Associations were evaluated within a retrospective cohort approach. All four loci were associated with ovarian cancer risk in BRCA2 carriers; rs10088218 per-allele hazard ratio (HR) = 0.81 (95% CI: 0.67-0.98) P-trend = 0.033, rs2665390 HR = 1.48 (95% CI: 1.21-1.83) P-trend = 1.8 × 10(-4), rs717852 HR = 1.25 (95% CI: 1.10-1.42) P-trend = 6.6 × 10(-4), rs9303542 HR = 1.16 (95% CI: 1.02-1.33) P-trend = 0.026. Two loci were associated with ovarian cancer risk in BRCA1 carriers; rs10088218 per-allele HR = 0.89 (95% CI: 0.81-0.99) P-trend = 0.029, rs2665390 HR = 1.25 (95% CI: 1.10-1.42) P-trend = 6.1 × 10(-4). The HR estimates for the remaining loci were consistent with odds ratio estimates for the general population. The identification of multiple loci modifying ovarian cancer risk may be useful for counseling women with BRCA1 and BRCA2 mutations regarding their risk of ovarian cancer., (© 2012 Wiley Periodicals, Inc.)

In this study, we present a novel complex rearrangement in the BRCA1 gene. The genomic rearrangement was identified using one of the two commercially available MLPA BRCA1 kits but was not confirmed with the other. In this report, we present the full characterization at the DNA and RNA levels of a new partial deletion of exon 20 of BRCA1. This is a complex deletion with four breakpoints which promotes aberrant splicing with partial deletion of exon 20 plus the insertion of a cryptic exon corresponding to a fragment of intron 20. The aberrant splicing generates an abnormal transcript with a frameshift that will result in a truncated BRCA1 protein.

Purposes: To review patients with a hematogenous and early post-surgical prosthetic joint infection (PJI) due to S. aureus treated with debridement and retention of the implant and to compare their clinical characteristics and outcome., Methods: From January 2000 all patients with a prosthetic joint infection treated in a single-center were prospectively registered and followed-up. All potentially variables associated with outcome were recorded. For the present study, cases with a hematogenous or early post-surgical PJI due to S. aureus treated with debridement and at least 2 years of follow-up were reviewed. Cox regression model to identify factors associated with outcome were applied., Results: 12 hematogenous and 53 early post-surgical PJI due to S. aureus were included. Number of patients presenting with fever, leucocyte count, C-reactive protein concentration, and the number of bacteremic patients were significantly higher in hematogenous infections while the number of polymicrobial infections was lower in hematogenous than in early post-surgical infections. The global failure rate in hematogenous and early post-surgical PJI was 58.7% and 24.5%, respectively (p=0.02). The Cox regression model identified hematogenous infections (OR: 2.57, CI95%: 1.02-6.51, p=0.04) and the need of a second debridement (OR: 4.61, CI95%: 1.86-11.4, p=0.001) as independent predictors of failure., Conclusion: Hematogenous infections were monomicrobial and had more severe symptoms and signs of infection than early post-surgical PJI. Hematogenous PJI due to S. aureus, using debridement with implant retention, had a worse outcome than early post-surgical infections.

Purpose: To evaluate the relationship between intraoperative cultures during hip arthroplasty, the different patient characteristics, and the risk of developing a prosthetic joint infection (PJI)., Methods: A prospective observational study was performed. Patients who underwent elective THA from March 2007 to March of 2011 were included. Three samples were taken just after arthrotomy: synovial fluid inoculated into blood culture flasks (SF), a tissue sample (TS), and a swab of peri- prosthetic tissue (S). Patients received standard antibiotic prophylaxis. The PJI rate within the first 3 months after arthroplasty was recorded., Results: 402 prostheses were included in the study. Contamination rate of synovial fluid was 10.2%. The most frequent isolated microorganism was coagulase-negative staphylococci (CoNS) (32 out of 41). Body mass index (BMI) was the only host characteristic associated with positive intraoperative culture (p=0.009). The PJI rate was 3.7%. Variables associated with PJI in the univariate and multivariate analysis were: age =67 years (p=0.012 OR: 5.35 (1.45-19.81); CI95%) and a BMI =35 (p=0.002, OR: 7.7 (2.12-27.85) CI95%). PJI rate among patients with BMI<35 with negative and positive intraoperative cultures was 3% and 2.7%, respectively, however, the rate among patients with BMI = 35 was 15% and 25%, respectively., Conclusion: A BMI =35 was associated with a higher risk of positive intraoperative culture during hip arthroplasty. In addition, a BMI ≥35 was independently associated with a high risk of PJI and the highest rate was documented among obese patients with positive intraoperative cultures.

Large genomic rearrangements are estimated to account for approximately 5-10% of all disease-causing mutations in BRCA1 and BRCA2 genes in patients with hereditary breast and ovarian cancer syndrome (HBOC). We use MRC-Holland Multiplex Ligation-dependent Probe Amplification (MLPA) to screen for such rearrangements in patients with HBOC and as a first step in our genetic testing workflow. The technique was applied to a set of 310 independent patients and detected eight different copy number alterations, corresponding to 2.6% of the studied samples. MLPA was also found to identify point mutations located in probe sequences. As commercial MLPA tests are not suitable for determining the specific breakpoints or for defining the exact extent of rearrangements, we applied a set of different complementary techniques to characterize these genetic alterations with greater precision. Long-range PCR amplification, RNA analysis, SNP-array chips, non-commercial MLPA probes, and FISH analysis were used to fully define the extent and mechanism of each alteration. In BRCA1, six rearrangements were characterized: deletion of E22, duplication of E9-E24, deletion of E16-E23, deletion of E1-E13, deletion of E1-E2 and duplication of E1-E2. In BRCA2, we studied a deletion of E15-E16 and a deletion of E1-E24. To the best of our knowledge, this is the most comprehensive study of the nature and underlying molecular causes of these mutational events in the BRCA1/2 genes.

Background: Socioeconomic level is a key determinant for health status. It may also influence on the utilization of hospital emergency services, although the factor responsible for that is not known. The main objective of the present report is to assess whether patients using frequently hospital emergency services have one or more variables related to low socioeconomic level., Patients and Methods: A standardized questionnaire was applied to 800 patients randomly selected from those attending the emergency services of a provincial reference hospital. It was gathered information on: income, education level, employment, loneliness, domestic violence, occupational accidents, and life style. Odds ratios (OR) and their 95% confidence intervals (CI) were estimated. Stepwise logistic regression analysis was used to ascertain the independent predictors for attending for than once to the emergency services., Results: In crude analysis, low income and low educational level were significantly related to the use of emergency services (both chi 2 for a trend < 0.05). Alcoholism (OR = 2.3, 95% CI = 1.2-4.7) and domestic violence (OR = 2.6, 95% CI = 0.9-8.1) were also related. Stepwise logistic regression analysis selected income (OR for intermediate level = 1.6, 95% CI = 1.0-2.7, and OR for low level = 2.3, 95% CI = 1.4-3.7) and alcoholism (OR = 2.9, 95% CI = 1.3-6.4) as independent predictors., Conclusions: Patients with low income and/or alcoholism use emergency hospital services more frequently.