[Patient-related stressful experiences in psychiatry residency]. / Patiëntgerelateerde stresserende ervaringen tijdens de opleiding psychiatrie.

BACKGROUND: Residents in psychiatry can be confronted with patient-related stressful experiences which can be impactful. Guidelines for dealing with these are lacking. AIM: Questioning residents in psychiatry about patient-related stressful adversities, their impact and resources to contribute to the existing literature. METHOD: Qualitative study through semi-structured interviews of six psychiatry residents, followed by data-analysis using thematic analysis. RESULTS: Reported patient-related stressful experiences were suicide, verbal and imminent physical aggression, questioning expertise, concerns about legal consequences, powerlessness and expectations. Cognitive and physical impact was experienced with symptoms similar to acute stress disorder. The most important resource is social support. CONCLUSION: In this study new patient related stressful experiences are reported that have not previously been described in the literature. Since estimating the impact of a patient related stressful experience proves difficult, it seems appropriate that the environment of the resident offers active support and further development of residents’ skills is stimulated.

Antimicrobial susceptibility profile of clinically relevant Bacteroides, Phocaeicola, Parabacteroides and Prevotella species, isolated by eight laboratories in the Netherlands.

OBJECTIVES: Recently, reports on antimicrobial-resistant Bacteroides and Prevotella isolates have increased in the Netherlands. This urged the need for a surveillance study on the antimicrobial susceptibility profile of Bacteroides, Phocaeicola, Parabacteroides and Prevotella isolates consecutively isolated from human clinical specimens at eight different Dutch laboratories. METHODS: Each laboratory collected 20-25 Bacteroides (including Phocaeicola and Parabacteroides) and 10-15 Prevotella isolates for 3 months. At the national reference laboratory, the MICs of amoxicillin, amoxicillin/clavulanic acid, piperacillin/tazobactam, meropenem, imipenem, metronidazole, clindamycin, tetracycline and moxifloxacin were determined using agar dilution. Isolates with a high MIC of metronidazole or a carbapenem, or harbouring cfiA, were subjected to WGS. RESULTS: Bacteroides thetaiotaomicron/faecis isolates had the highest MIC90 values, whereas Bacteroides fragilis had the lowest MIC90 values for amoxicillin/clavulanic acid, piperacillin/tazobactam, meropenem, imipenem and moxifloxacin. The antimicrobial profiles of the different Prevotella species were similar, except for amoxicillin, for which the MIC50 ranged from 0.125 to 16 mg/L for Prevotella bivia and Prevotella buccae, respectively. Three isolates with high metronidazole MICs were sequenced, of which one Bacteroides thetaiotaomicron isolate harboured a plasmid-located nimE gene and a Prevotella melaninogenica isolate harboured a nimA gene chromosomally.Five Bacteroides isolates harboured a cfiA gene and three had an IS element upstream, resulting in high MICs of carbapenems. The other two isolates harboured no IS element upstream of the cfiA gene and had low MICs of carbapenems. CONCLUSIONS: Variations in resistance between species were observed. To combat emerging resistance in anaerobes, monitoring resistance and conducting surveillance are essential.

Colistin-resistant Enterobacterales among veterinary healthcare workers and in the Dutch population.

Objectives: Plasmid-mediated colistin resistance can be transferred from animals to humans. We investigated the prevalence of carriage of mcr-mediated colistin-resistant Escherichia coli and Klebsiella pneumoniae (ColR-E/K) in veterinary healthcare workers and in the general population in the Netherlands. Methods: Two cross-sectional population studies were performed: one among veterinary healthcare workers and one in the general population. Participants sent in a faecal sample and filled in a questionnaire. Samples were analysed using selective enrichment and culture. Mobile colistin resistance genes (mcr) were detected by PCR and ColR-E/K were sequenced using Illumina and Nanopore technologies. Results: The prevalence of mcr-mediated ColR-E/K was 0.2% (1/482, 95% CI 0.04%-1.17%) among veterinary personnel and 0.8% (5/660, 95% CI 0.3%-1.8%) in the population sample. mcr-1 was found in E. coli from four persons, mcr-8 in K. pneumoniae from one person and another person carried both mcr-1 and mcr-8 in a K. pneumoniae isolate. mcr-1 was found on different plasmid types (IncX4, IncI1 and IncI2), while mcr-8 was found on IncF plasmids only. Conclusions: mcr-mediated ColR-E/K resistance was uncommon in both populations. Professional contact with animals does not increase the chance of carriage of these bacteria in the Netherlands at present. mcr-8 was found for the first time in the Netherlands. Surveillance of colistin resistance and its underlying mechanisms in humans, livestock and food is important in order to identify emerging trends in time.

The multilayered structure of the human corpus spongiosum.

PURPOSE: Urethral reconstruction is performed in patients with urethral strictures or for correction of congenital disorders. In the case of shortage of tissue, engineered tissue may enhance urethral reconstruction. As the corpus spongiosum (CS) is important in supporting the function of the urethra, tissue engineering of the urethra should be combined with reconstruction of a CS. For that purpose, detailed knowledge of the composition of the CS, more specifically its extracellular matrix (ECM) and vascularization is needed for scaffold design. The objective of this study is to analyze the microarchitecture of the CS through (immuno) histology and scanning electron microscopy (SEM). METHODS: The CS including the urethra of patients undergoing male-to-female genital confirming surgery was harvested. This CS was fixed and processed for either (immuno) histology or for SEM. RESULTS: Four layers could be distinguished in the CS; first a transition zone from urethra epithelium to a collagen rich layer, which was highly vascularized, followed by a second, elastin rich layer. The third layer was formed by veins, arteries and vascular spaces and the last layer showed the transition from this vascular rich region to the collagen rich tunica albuginea. In this layer collagen bundles intertwined with elastic fibres. In the CS different components of the ECM were visible and distinguishable. CONCLUSION: This study provides novel and detailed information on the microarchitecture of the CS and the distribution of vascularization, which is important for scaffold design in tissue engineering.

A new protocol for evaluating the efficacy of some dispensing systems of a packaging in the microbial protection of water-based preservative-free cosmetic products.

OBJECTIVE: A new protocol is described for assessing the efficacy of the dispenser of some packaging systems (PSs) of preservative-free cosmetic products in protecting both their contained formula and their delivered doses. METHODS: Practically, aiming at mimicking contacts with a non-sterile skin or fingers, the dispensing system is put into contact with a pre-contaminated fabric by a standardized colonization of P. aeruginosa. RESULTS: When applied to three different types of packaging, results show clear differences in both criteria between these conditioning articles, that is variable efficacies in protecting the contained product and the delivered doses, knowing that the first aspect is of paramount importance. CONCLUSION: The proposed protocol is proved being able to discriminate between different PSs and provides information on strong and weak features of certain types dispensing technologies prone to efficiently decrease either the dose contamination or to prevent contamination in reaching the contained product. Therefore, the proposed protocol can contribute to an objective selection of a PS for protecting a cosmetic care product with a low content of preservative or preservative free.

Two Novel Mutations in the SLC25A4 Gene in a Patient with Mitochondrial Myopathy.

In a 28-year-old male with a mild mitochondrial myopathy manifesting as exercise intolerance and early signs of cardiomyopathy without muscle weakness or ophthalmoplegia, we identified two novel mutations in the SLC25A4 gene: c.707G>C in exon 3 (p.(R236P)) and c.116_137del in exon 2 (p.(Q39Lfs*14)). Serum lactate levels at rest were elevated (12.7 mM). Both the patient's father and brother were heterozygous carriers of the c.707G>C mutation and were asymptomatic. The second mutation causes a 22 bp deletion leading to a frame shift likely giving rise to a premature stop codon and nonsense-mediated decay (NMD). The segregation of the mutations could not be tested directly as the mother had died before. However, indirect evidence from NMD experiments showed that the two mutations were situated on two different alleles in the patient. This case is unique compared to other previously reported patients with either progressive external ophthalmoplegia (PEO) or clear hypertrophic cardiomyopathy with exercise intolerance and/or muscle weakness carrying recessive mutations leading to a complete absence of the SLC25A4 protein. Most likely in our patient, although severely reduced, SLC25A4 is still partially present and functional.

PGD and heteroplasmic mitochondrial DNA point mutations: a systematic review estimating the chance of healthy offspring.

BACKGROUND: Mitochondrial disorders are often fatal multisystem disorders, partially caused by heteroplasmic mitochondrial DNA (mtDNA) point mutations. Prenatal diagnosis is generally not possible for these maternally inherited mutations because of extensive variation in mutation load among embryos and the inability to accurately predict the clinical expression. The aim of this study is to investigate if PGD could be a better alternative, by investigating the existence of a minimal mutation level below which the chance of an embryo being affected is acceptably low, irrespective of the mtDNA mutation. METHODS: We performed a systematic review of muscle mutation levels, evaluating 159 different heteroplasmic mtDNA point mutations derived from 327 unrelated patients or pedigrees, and reviewed three overrepresented mtDNA mutations (m.3243A>G, m.8344A>G and m.8993T>C/G) separately. RESULTS: Mutation levels were included for familial mtDNA point mutations only, covering all affected (n = 195) and unaffected maternal relatives (n = 19) from 137 pedigrees. Mean muscle mutation levels were comparable between probands and affected maternal relatives, and between affected individuals with tRNA- versus protein-coding mutations. Using an estimated a priori prevalence of being affected in pedigrees of 0.477, we calculated that a 95% or higher chance of being unaffected was associated with a muscle mutation level of 18% or less. At a mutation level of 18%, the predicted probability of being affected is 0.00744. The chance of being unaffected was lower only for the m.3243A>G mutation (P < 0.001). Most carriers of mtDNA mutations will have oocytes with mutation levels below this threshold. CONCLUSIONS: Our data show, for the first time, that carriers of heteroplasmic mtDNA mutations will have a fair chance of having healthy offspring, by applying PGD. Nevertheless, our conclusions are partly based on estimations and, as indicated, do not provide absolute certainty. Carriers of mtDNA should be informed about these constraints.

Large scale mtDNA sequencing reveals sequence and functional conservation as major determinants of homoplasmic mtDNA variant distribution.

The mitochondrial DNA (mtDNA) is highly variable, containing large numbers of pathogenic mutations and neutral polymorphisms. The spectrum of homoplasmic mtDNA variation was characterized in 730 subjects and compared with known pathogenic sites. The frequency and distribution of variants in protein coding genes were inversely correlated with conservation at the amino acid level. Analysis of tRNA secondary structures indicated a preference of variants for the loops and some acceptor stem positions. This comprehensive overview of mtDNA variants distinguishes between regions and positions which are likely not critical, mainly conserved regions with pathogenic mutations and essential regions containing no mutations at all.

Inherited mitochondrial variants are not a major cause of age-related hearing impairment in the European population.

Mitochondrial DNA (mtDNA) mutations have been implicated in various age-related diseases. To further clarify the role of mtDNA variants in age-related hearing impairment (ARHI), we determined the DNA sequence of the entire mitochondrial genome of 400 individuals using the Affymetrix Human Mitochondrial Resequencing Array. These were the 200 worst hearing and the 200 best hearing from a collection of 947 Belgian samples. We performed association tests with individual mitochondrial variants, comparison of the mutation load, and association with European haplogroups and their interaction with environmental risk factors. We also tested the influence of rare variants on ARHI. None of these tests showed any association with ARHI.

Critical evaluation of the EU-technical guidance on shelf-life studies for L. monocytogenes on RTE-foods: a case study for smoked salmon.

In November 2008, a technical guidance document on the challenge test protocol was published by the EU CRL (Community of Reference Laboratory) for L. monocytogenes. This document describes the practical aspects on the execution of a challenge test in order to comply to the EU Commission regulation N° 2073/2005 on microbiological criteria for foodstuff. In this guideline two approaches are specified. On the one hand challenge tests, based on actual data measurements at the beginning and end of the shelf-life of products stored under reasonably foreseen T-profile, are described. On the other hand, growth potential is calculated by predictive models using a validated maximum specific growth rate. The present study evaluates the two above mentioned approaches on cold smoked salmon, a typical risk product for L. monocytogenes. The focus is on: (i) the relative importance of intrabatch versus interbatch variability, (ii) the concept of a simple challenge test based on actual data at start and end of shelf life versus a modelling approach and (iii) the interpretation of challenge tests. Next to this, available tertiary models were used to estimate the growth potential of these products based on their initial physicochemical characteristics. From the results it could be concluded that in some batches considerable intrabatch variability was obtained. In general, however, the interbatch variability was significantly higher than intrabatch variability. Concerning the two above mentioned methods for challenge tests, it can be stated that the first approach (simple challenge test) can be set up rather rapidly and is cost-effective for SMEs (small and medium enterprises) but provides only a single isolated outcome. This implies that challenge tests should be redone if changes occur in composition or production process. The second (modelling) approach, using extended challenge tests to establish growth parameters needs larger set ups and more complicated data analysis, which makes them more expensive. Using available tertiary models has the major advantage that the most important intrinsic and extrinsic factors can be included for the prediction of the growth parameter. It was clear that product specific models, taking into account the interaction effects with background flora, performed the best. Regarding the challenge tests, it can be concluded that the best approach to choose will depend on the particular context as in the end both approaches will lead to the same conclusion.

Defective complex I assembly due to C20orf7 mutations as a new cause of Leigh syndrome.

BACKGROUND: Leigh syndrome is an early onset, progressive, neurodegenerative disorder with developmental and motor skills regression. Characteristic magnetic resonance imaging abnormalities consist of focal bilateral lesions in the basal ganglia and/or the brainstem. The main cause is a deficiency in oxidative phosphorylation due to mutations in an mtDNA or nuclear oxidative phosphorylation gene. METHODS AND RESULTS: A consanguineous Moroccan family with Leigh syndrome comprise 11 children, three of which are affected. Marker analysis revealed a homozygous region of 11.5 Mb on chromosome 20, containing 111 genes. Eight possible mitochondrial candidate genes were sequenced. Patients were homozygous for an unclassified variant (p.P193L) in the cardiolipin synthase gene (CRLS1). As this variant was present in 20% of a Moroccan control population and enzyme activity was only reduced to 50%, this could not explain the rare clinical phenotype in our family. Patients were also homozygous for an amino acid substitution (p.L159F) in C20orf7, a new complex I assembly factor. Parents were heterozygous and unaffected sibs heterozygous or homozygous wild type. The mutation affects the predicted S-adenosylmethionine (SAM) dependent methyltransferase domain of C20orf7, possibly involved in methylation of NDUFB3 during the assembly process. Blue native gel electrophoresis showed an altered complex I assembly with only 30-40% of mature complex I present in patients and 70-90% in carriers. CONCLUSIONS: A new cause of Leigh syndrome can be a defect in early complex I assembly due to C20orf7 mutations.

The unfolding clinical spectrum of POLG mutations.

BACKGROUND: Mutations in the DNA polymerase-gamma (POLG) gene are a major cause of clinically heterogeneous mitochondrial diseases, associated with mtDNA depletion and multiple deletions. OBJECTIVE: To determine the spectrum of POLG mutations in our Dutch patient cohort, to evaluate the pathogenicity of novel mutations, and to establish genotype-phenotype correlations. RESULTS: The authors identified 64 predominantly recessive mutations in 37 patients from a total of 232 patients, consisting of 23 different mutations. The substitution p.A467T was most frequently observed (n = 23), but was as frequent in childhood cases as in adult cases. Five new pathogenic recessive mutations, p.Lys925ArgfsX42, p.R275X, p.G426S, p.A804T and p.R869Q were identified. The known dominant chronic progressive external ophthalmoplegia (CPEO) mutation p.R943H was for the first time associated with premature ovarian failure as well. In 19 patients the authors identified only a single recessive mutation, or a sequence variant with unclear clinical significance. The data substantiate earlier observations that in POLG patients a fatal status epilepticus and liver failure can be triggered by sodium valproate. It is therefore important to exclude POLG mutations before administering this treatment. CONCLUSION: The clinical features of the patient are the most important features to select putative POLG mutation carriers and not the presence of mtDNA deletions or OXPHOS (oxidative phosphorylation) activity. The authors conclude that POLG mutations are an important cause of heterogeneous mitochondrial pathology and that more accurate genotype-phenotype correlations allow a more rapid genetic diagnosis and improved prognosis for mutation carriers.

Mutations in the ND5 subunit of complex I of the mitochondrial DNA are a frequent cause of oxidative phosphorylation disease.

BACKGROUND: Detection of mutations in the mitochondrial DNA (mtDNA) is usually limited to common mutations and the transfer RNA genes. However, mutations in other mtDNA regions can be an important cause of oxidative phosphorylation (OXPHOS) disease as well. OBJECTIVE: To investigate whether regions in the mtDNA are preferentially mutated in patients with OXPHOS disease. METHODS: Screening of the mtDNA for heteroplasmic mutations was performed by denaturing high-performance liquid chromatography analysis of 116 patients with OXPHOS disease but without the common mtDNA mutations. RESULTS: An mtDNA sequence variant was detected in 15 patients, 5 of which were present in the ND5 gene. One sequence variant was new and three were known, one of which was found twice. The novel sequence variant m.13511A-->T occurred in a patient with a Leigh-like syndrome. The known mutation m.13513G-->A, associated with mitochondrial encephalomyopathy lactic acidosis and stroke-like syndrome (MELAS) and MELAS/Leigh/Leber hereditary optic neuropathy overlap syndrome, was found in a relatively low percentage in two patients from two different families, one with a MELAS/Leigh phenotype and one with a MELAS/chronic progressive external ophthalmoplegia phenotype. The known mutation m.13042G-->A, detected previously in a patient with a MELAS/myoclonic epilepsy, ragged red fibres phenotype and in a family with a prevalent ocular phenotype, was now found in a patient with a Leigh-like phenotype. The sequence variant m.12622G-->A was reported once in a control database as a polymorphism, but is reported in this paper as heteroplasmic in three brothers, all with infantile encephalopathy (Leigh syndrome) fatal within the first 15 days of life. Therefore, a causal relationship between the presence of this sequence variant and the onset of mitochondrial disease cannot be entirely excluded at this moment. CONCLUSIONS: Mutation screening of the ND5 gene is advised for routine diagnostics of patients with OXPHOS disease, especially for those with MELAS- and Leigh-like syndrome with a complex I deficiency.

Increased risk for cardiorespiratory failure associated with the A3302G mutation in the mitochondrial DNA encoded tRNALeu(UUR) gene.

Screening the mitochondrial DNA of a 64-year-old woman with mitochondrial myopathy revealed 76% of the tRNA(Leu(UUR)) A3302G mutation in muscle. Muscle of her affected son carried 96% mutated mitochondrial DNA. Both patients were biopsied twice, showing isolated complex I deficiency in the son's first biopsy, additional increased (within normal range) complex II + III activities in his second biopsy, combined complex I, II + III deficiency in mothers first biopsy and additional complex IV deficiency in her second biopsy. After a stay in the mountains, the son died of cardiac arrhythmia. The A3302G mutation has been reported before and is associated with mitochondrial myopathy and cardiorespiratory failure. Pathogenesis is explained by abnormal mtRNA processing, which was also reported for the adjacent C3303T mutation associated with cardiomyopathy and/or skeletal myopathy. Our findings suggest that a high mutation load of the A3302G mutation can lead to fatal cardiorespiratory failure, likely triggered by low environmental oxygen pressure and exercise.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on fatty acid availability and neural tube formation in cynomolgus macaque, Macaca fascicularis.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is known to alter carbohydrate utilization and specific steps in lipid metabolism. TCDD interacts with estradiol in mobilizing specific fatty acids in chickens that may be a cause of cranial/beak malformations in this species. This study was designed to test the hypothesis that TCDD simultaneously alters critical fatty acid mobilization during early pregnancy and determine if those changes correlate to morphological defects of the developing neural tube in the nonhuman primate. Cynomolgus macaques were treated with a single dose of 4 microg/kg body weight (BW) TCDD on gestational day 15 or 20. Pregnancies were terminated by hysterectomy on gestational day 24-26 and embryos were examined to determine morphology of the developing neural tube. Maternal blood samples were used for fatty acid quantification. Embryos exhibited cellular changes, mainly increased cell death, and intercellular spaces in the neural tube, suggestive of an adverse effect on the developing nervous system. Significant decreases on fatty acid composition were found on some of the eight classes of lipids analyzed. Particularly, a decrease was observed in the n-3 (40-60%) and n-6 (47-75%) essential fatty acids in treated pregnancies compared to untreated controls. These data demonstrate the effect of TCDD in decreasing maternal levels of n-3 and n-6 fatty acids that are considered necessary for normal development in mammals. Since neural tube development is dependent, in part, on n-3 and n-6 fatty acids, it is possible that the limitation of these essential fatty acids in plasma resulted in the observed detrimental effects on early brain development.

Do anal fistulas in Crohn's disease behave differently and defy Goodsall's rule more frequently than fistulas that are cryptoglandular in origin?

OBJECTIVES: The complexity of anal fistulas is different in patients with and without Crohn's disease and in men and women. This may affect the localization of the internal orifice. We compared the characteristics of anal fistulas and the accuracy of Goodsall's rule in predicting the position of the internal orifice in male and female Crohn's and non-Crohn's patients. METHODS: A total of 191 fistula tracks in 182 consecutive patients (110 men and 72 women) were analyzed prospectively. Of the patients, 63 were diagnosed with Crohn's disease. The positions of the orifices were recorded and the accuracy of Goodsall's rule determined. RESULTS: The distribution of fistula subtypes among Crohn's and non-Crohn's patients differed significantly (p = 0.0471). Fistulas with an anterior external opening occurred more frequently in Crohn's patients (p = 0.0350) and in women (p = 0.0030). Fistulas with a posterior external orifice were observed more frequently in non-Crohn's patients (p = 0.0350) and in men (p = 0.0028). Overall, Goodsall's rule performed less well in women compared with men (p = 0.0633). The accuracy of Goodsall's rule overall was not affected by Crohn's disease. In female non-Crohn's patients, the positive predictive value of a posterior external orifice was lower than in men (p = 0.0406). CONCLUSIONS: The distribution of fistula subtypes and the ratio of anterior and posterior external openings among Crohn's and non-Crohn's patients differ significantly. Many fistulas defy Goodsall's rule, particularly in women and when applied to fistulas with anterior external orifices. The popular rule, however, falls equally short in Crohn's and non-Crohn's fistulas.

The nonhuman primate as a model of developmental immunotoxicity.

Macaques are well suited for preclinical testing of biopharmaceutics due to reproductive and developmental similarities with humans. In order to characterize ontogeny of the immune system in this model, we studied lymphocyte and antigen-presenting cell populations in developing lymphoid tissues of rhesus macaque fetuses during the second and third trimesters [gestation days (GD) 75-145, term 165 days]. Systemic lymphoid tissues (thymus, spleen and lymph nodes, and intestinal tissue) were examined for morphology and cell surface markers by immunohistochemistry. Lymphocytes were further characterized by flow cytometry for differentiation markers. Splenic tissue from early second trimester fetuses was populated mainly by CD20+ B cells while the thymus contained large numbers of CD3+ T cells. In the late second trimester (day 80), approximately equal populations of B and T cells were present in both tissues and numerous dendritic cells (p55+) were present in the intestinal lamina propria. By the second trimester, the rhesus macaque fetal lymphoid system is well developed. Analysis of lymphoid organs from retinoic acid-treated fetuses indicated that the T-cell (thymus)-dependent compartment of the spleen white pulp in specimens with thymic aplasia showed a reduction in size and proportion of CD3+ T cells compared to controls. Our findings indicate that RA-induced thymic defects result in disrupted development of the splenic T-cell-dependent compartment.

Exposure of cynomolgus monkey embryos to retinoic acid causes thymic defects: effects on peripheral lymphoid organ development.

We have previously reported that exposure of monkey embryos to 13-cis-retinoic acid (cRA) results in thymic defects. In this study, we analyzed lymphocyte and antigen-presenting cell populations at gestational days (GDs) 80-100 in the thymus, spleen, mesenteric lymph nodes, and gut-associated lymphoid tissue following a teratogenic dosing regimen of cRA (2.5 and 5 mg/kg) at GD14-27. Tissue sections were immunostained for T-cells (anti-CD3), B-cells (anti-CD20), dendritic cells (p55), and major histocompatibility class II (anti-HLA-DR). Digital images of spleen sections were analyzed to obtain the relative area occupied by the cell subsets within the white pulp (WP). Compared with controls, the T-cell dependent compartment of the spleen WP in specimens with perturbed thymic development (aplasia and severe hypoplasia) showed a reduction in size and proportion of CD3(+) T cells. Our findings indicate that cRA-induced thymic defects result in disrupted development of the splenic T-cell dependent compartment.