A Multiplex PCR and DNA-Sequencing Workflow on Serum for the Diagnosis and Species Identification for Invasive Aspergillosis and Mucormycosis.

There has been significant increase in the use of molecular tools for the diagnosis of invasive aspergillosis (IA) and mucormycosis. However, their range of detection may be too limited as species diversity and coinfections are increasing. Here, we aimed to evaluate a molecular workflow based on a new multiplex PCR assay detecting the whole Aspergillus genus and the Mucorales order followed by a species-specific PCR or a DNA-sequencing approach for IA and/or mucormycosis diagnosis and species identification on serum. Performances of the MycoGENIE Aspergillus spp./Mucorales spp. duplex PCR kit were analyzed on a broad range of fungal strains and on sera from high-risk patients prospectively over a 12-month period. The kit allowed the detection of nine Aspergillus species and 10 Mucorales (eight genera) strains assessed. No cross-reactions between the two targets were observed. Sera from 744 patients were prospectively analyzed, including 35 IA, 16 mucormycosis, and four coinfections. Sensitivity varies from 85.7% (18/21) in probable/proven IA to 28.6% (4/14) in COVID-19-associated pulmonary aspergillosis. PCR-positive samples corresponded to 21 A. fumigatus, one A. flavus, and one A. nidulans infections. All the disseminated mucormycosis were positive in serum (14/14), including the four Aspergillus coinfections, but sensitivity fell to 33.3% (2/6) in localized forms. DNA sequencing allowed Mucorales identification in serum in 15 patients. Remarkably, the most frequent species identified was Rhizomucor pusillus (eight cases), whereas it is barely found in fungal culture. This molecular workflow is a promising approach to improve IA and mucormycosis diagnosis and epidemiology.

Diagnosis of monogenic liver diseases in childhood by next-generation sequencing.

Next-generation sequencing (NGS) has opened up novel diagnostic opportunities for children with unidentified, but suspected inherited diseases. We describe our single-center experience with NGS diagnostics in standard clinical scenarios in pediatric hepatology. We investigated 135 children with suspected inherited hepatopathies, where initially no causative pathogenic variant had been identified, with an amplicon-based NGS panel of 21 genes associated with acute and chronic hepatopathies. In 23 of these patients, we detected pathogenic or likely pathogenic variants in 10 different genes. We present 6 novel variants. A total of 14 of these patients presented with the characteristic phenotype of the related hepatopathy. Nine patients showed only few or atypical clinical symptoms or presented with additional signs. In another 13 out of 135 cases, we detected variants of unknown significance (VUS) in 9 different genes. Only 2 of these patients showed characteristic phenotypes conclusive with the detected variants, whereas 11 patients showed unspecific or atypical phenotypes. Our multi-gene panel is a fast and comprehensive tool to diagnose inherited pediatric hepatopathies. We also illustrate the challenge of dealing with genetic variants and highlight arising clinical questions, especially in patients with atypical phenotypes.

[Wilson's disease : What has been confirmed in diagnostic and therapy?] / Morbus Wilson : Was ist gesichert in Diagnostik und der Therapie?

Wilson's disease (WD) is a rare autosomal recessive disorder characterized by abnormal copper accumulation. Presenting a broad variety of phenotypes and, thus, being a chameleon within the group of metabolic diseases, the manifold clinical symptoms of WD can include hepatologic, neurologic, and psychiatric manifestations. Early onset presentations in infancy and late-onset manifestations in adults older than 70 years of age have been described. If the typical laboratory blood test values are missing, the diagnosis of WD may be difficult and often involves a combination of different parameters. Novel test methods like the identification of the relative exchangeable copper have not been validated within a sufficient cohort of WD patients as of yet and therefore do not currently play a crucial role within the clinical setting. Consequently any patient with reasonable suspected diagnosis of WD needs to be presented to a (pediatric) gastroenterologist and/or (pediatric) neurologist. Different medical treatments including drugs such as copper chelating agents are commonly used in the clinical setting. Liver transplantation may be the ultima ratio in selected patients. Dietary changes involving a low copper diet play only a minor role. Due to the fact the use of tetrathiomolybdate is still not approved, the treatment of advanced and progressive neurologic symptoms remains a major challenge. In any case, life-long medical supervision and treatment governed by a specialist is absolutely essential. Early diagnosis and early and life-long treatment lead to better prognoses and do not negatively influence the overall life expectancy.

"Current state and prospects in managing liver transplanted children".

Pediatric liver transplantation (LTx) has revolutionized life chances and perspectives of children with liver disease. Following rapid establishment of the therapeutic concept in the early years of pediatric transplant medicine, more aspects beyond plain survival become increasingly important. In addition to improving the short to medium-term survival rates, researchers are focusing on themes such as rehabilitation, adherence and quality of life, long-term graft fibrosis and dysfunction, as well as the consequences of long-term immunosuppression. Also, more protocol biopsy data are available to evaluate increasing graft fibrosis. To manage their conditions, patients will need access to highly experienced pediatric liver transplant centers where clinical research will examine modulators of renal disease, endocrine and cardiovascular comorbidity and the development of graft fibrosis and malignancies. Assessment and evaluation of health-related quality of life and factors which influence clinical tolerance, adherence and transition from child to adult care will also be investigated. The analysis of multi-national registry data and more than 40years of experience with large patient cohorts will provide important clues to treatment and will thus get increasing attention. In the future, longitudinal assessment of the outcome for pediatric LTx patients should include more functional aspects than plain survival rates or laboratory parameters.

Prognostic marker for liver disease due to alpha1-antitrypsin deficiency.

BACKGROUND: Only some Alpha1-antitrypsin deficiency (A1ATD) PiZZ patients develop liver cirrhosis and portal hypertension. Aim of the study was to investigate the course of liver disease associated with PiZZ A1ATD and to determine prognostic factors. PATIENTS: We retrospectively reviewed the clinical and laboratory data of all PiZZ children up to 18 years of age admitted to our centre since 1978. 53 patients (age at first visit 2 days to 12 years) met our criteria. METHODS: The children were divided into 2 groups: group 1 'bad prognosis', meaning the patients which were on the waiting list for liver transplantation (LTx), had a liver transplantation or had died, and group 2 'good prognosis', containing the patients they were living with their own liver. We analysed family history including smoking, gestational age, maternal age at delivery, date of birth, sex, neonatal history, breast-feeding, symptoms at presentation, clinical and laboratory data and date of LTx and/or death. RESULTS: Various anamnesis parameters such as manifestation of neonatal cholestasis showed no prognostic significance. In contrast the laboratory parameters thrombocytes (p=0.008), bilirubin (p<0.001), prothrombin time (p<0.001), choline-sterase (p<0.001), gamma-GT (p=0.001) and GOT (p=0.002) showed a correlation with a liver transplantation and/or death. CONCLUSION: Prognosis is difficult to determine at an early stage of this disease, but various laboratory parameters can help to predict an outcome. Therefore a regular follow-up is necessary for the children.

Biliary atresia: lessons learned from the voluntary German registry.

INTRODUCTION: Aim of the study was to carry out a 5-year survey of German patients with biliary atresia (BA) and to launch a discussion regarding the feasibility of voluntary registries in unregulated healthcare systems. METHODS: A retrospective analysis of German BA patients born between 2001 and 2005, based on data collected from the voluntary European Biliary Atresia Registry (EBAR), was carried out and supplemented by data from all BA patients who underwent liver transplantation at the only 4 pediatric transplantation centers (pLTx) in Germany which are so far not registered at EBAR. Survival rates were calculated using Kaplan-Meier analysis and compared by Cox regression to determine the predictive value of age at surgery and the influence of the center size (fewer or more than 5 patients/study period) on overall survival and survival with native liver. RESULTS: A critical review of the 148 German EBAR charts revealed that 11 patients (7.4%) had no biliary atresia. The remaining 137 patients from EBAR together with 46 BA patients who underwent LTx without prior registration at EBAR were evaluated with a median follow-up of 39 months (range: 25-85 months). 29 hospitals performed a total of 159 Kasai procedures, but only 7 centers treated 5 or more patients (116 patients, range: 5-68), and 22 hospitals performed less than 5 KP (43 patients, range: 1-4). Primary LTx was performed in 21 patients (11.5%) and 3 patients died without surgical intervention. 16 patients were lost to follow-up (8.7%). Overall survival after 2 years was 83.3% (139 patients), including 105 patients (63%) who had undergone LTx and 34 patients (20.3%) with native liver. 28 patients died (16.7%), 8 after LTx (5.8%). The experience of the center was the only factor with a significant predictive value for jaundice-free survival with native liver (p=0.001). CONCLUSION: 25% of all German BA patients were not registered at EBAR, and 29 clinics were involved in the surgical management of BA patients. Therefore a new approach consisting of an internet-based decentralized registry for rare neonatal liver diseases is outlined which could improve the future management of patients with BA. The centralization of such patients at experienced centers with higher caseloads is necessary in Germany and would improve the outcome of patients with biliary atresia.

[Predictive parameters in children with biliary atresia]. / Prognostische Marker bei Kindern mit Gallengangatresie.

Most children with biliary atresia require liver transplantation, and only about 20% survive in the long term with their native livers. Prognostic factors that determine disease progression are still lacking. This retrospective survey of 85 BA patients from 1993 to 2003 was aimed to evaluate prognostic factors using the log rank test. After 5 years 40% of the patients are alive with their native livers (35/85), 26 of them with normal bilirubin (31%). Age at Kasai operation (P=0.46), degree of liver fibrosis (P=0.95), and all laboratory test results before Kasai failed to correlate with outcome. Normal levels of bilirubin 3, 6, and 12 months after Kasai and of aspartate aminotransferase with gammaGT after 6 months are associated with survival with native liver. In conclusion our data demonstrate that a lack of predictive factors must prevent primary liver transplantation in BA patients.

[Results of a cardiovascular disease prevention study among administrative employees of a large city]. / Ergebnisse einer Herz-Kreislauf-Präventionstudie bei Angestellten einer Grossstadtverwaltung.

In parallel with the demographic changes in an increasingly ageing German population, cardiovascular diseases (HKE) are becoming increasingly frequent and thus more and more important. The main aim of this work is to derive individual preventive measures on the basis of classic risk factors. This study was performed on 101 administrative employees of a large city (71 women and 30 men). The tests included a general investigation of medical status [current individual and family case history, body mass index (BMI)] as well as the determination of the following laboratory values: glucose, HDL and LDL cholesterol. In addition, the study was completed by a job analysis, including a survey of individual health behaviour. Referring to the PROCAM study, ten traditional risk factors for cardiovascular disease (arterial blood pressure >140/90 mmHg, BMI > or =25 kg/m (2), family disposition etc.) were probed. On the basis of these results individual risk profiles for each participant were rated. A vulnerable person received individually tailored prevention recommendations, which were adapted to the individual health behaviour of the subjects. Each of the 101 study participants received a personal results and prevention bulletin with individual test results and prevention suggestions. This high effort promises a better implementation as general advice for prevention in terms of classic risk factors.

European biliary atresia registries: summary of a symposium.

Biliary atresia (BA) is a rare but potentially devastating disease. The European Biliary Atresia Registry (EBAR) was set up to improve data collection and to develop a pan-national and interdisciplinary strategy to improve clinical outcomes. From 2001 to 2005, 100 centers from 22 countries registered with EBAR via its website (www.biliary-atresia.com). In June 2006, the first meeting was held to evaluate results and launch further initiatives. During a 5-year period, 60 centers from 19 European countries and Israel sent completed registration forms for a total of 514 BA patients. Assuming the estimated incidence of BA in Europe is 1:18,000 live births, 35% of the expected 1488 patients from all EBAR participating countries were captured, suggesting that reporting arrangements need improvement. At the meeting, the cumulative evaluation of 928 BA patients including patients from other registries with variable follow-up revealed an overall survival of 78% (range from 41% to 92%), of whom 342 patients (37%) have had liver transplants. Survival with native liver ranged from 14% to 75%. There was a marked variance in reported management and outcome by country (e.g., referral patterns, timing of surgery, centralization of surgery). In conclusion, EBAR represents the first attempt at an overall evaluation of the outcome of BA from a pan-European perspective. The natural history and outcome of biliary atresia is of considerable relevance to a European population. It is essential that there is further support for a pan-European registry with coordination of clinical standards, further participation of parent support groups, and implementation of online data entry and multidisciplinary clinical and basic research projects.

Therapeutic silencing of mutant huntingtin with siRNA attenuates striatal and cortical neuropathology and behavioral deficits.

Huntington's disease (HD) is a neurodegenerative disorder caused by expansion of a CAG repeat in the huntingtin (Htt) gene. HD is autosomal dominant and, in theory, amenable to therapeutic RNA silencing. We introduced cholesterol-conjugated small interfering RNA duplexes (cc-siRNA) targeting human Htt mRNA (siRNA-Htt) into mouse striata that also received adeno-associated virus containing either expanded (100 CAG) or wild-type (18 CAG) Htt cDNA encoding huntingtin (Htt) 1-400. Adeno-associated virus delivery to striatum and overlying cortex of the mutant Htt gene, but not the wild type, produced neuropathology and motor deficits. Treatment with cc-siRNA-Htt in mice with mutant Htt prolonged survival of striatal neurons, reduced neuropil aggregates, diminished inclusion size, and lowered the frequency of clasping and footslips on balance beam. cc-siRNA-Htt was designed to target human wild-type and mutant Htt and decreased levels of both in the striatum. Our findings indicate that a single administration into the adult striatum of an siRNA targeting Htt can silence mutant Htt, attenuate neuronal pathology, and delay the abnormal behavioral phenotype observed in a rapid-onset, viral transgenic mouse model of HD.

Psychological effects of occupational exposure to organic solvent mixtures on printers.

PURPOSE: Neuropsychiatric symptoms associated with long-term exposure to organic solvents have been investigated for a long time in many occupational studies. The aim of our study was to identify preclinical effects of low-level chronic solvent exposure on the cognitive performance of silk-screen printers. METHODS: A cross-sectional study was conducted including 38 printers which were exposed to organic solvent mixtures and 45 controls without neurotoxic exposure. The psychometric test battery included nine tests and two questionnaires. RESULTS: The sum of scales or the total score of the PNF was also significantly (p <0.001) higher in the exposed group than in the non-exposed. The printers performed worse in some psychometric tests than did controls. Cross validation with the psychometric performances resulted in 69.6% of participants being correctly classified (55.9% printers and 80% controls). Highly significant differences (p<0.001) between both groups of exposed and non-exposed workers were found for the mean values of the multidimensional psychometric score. CONCLUSIONS: These results of our study proved not only that chronic exposure to organic solvent mixtures could induce a psychological effect but also that the psychological/psychometrical tests performed in this study were capable of predicting neurotoxicity.

[Influence of long term occupational exposure to solvents on colour vision]. / Einfluss langjähriger beruflicher Lösemittelexposition auf das Farbensehen.

BACKGROUND: The study was designed to determine the influence of chronic occupational exposition of organic solvent mixtures on colour vision of car painters. SUBJECTS AND METHODS: The 123 subjects (2 groups differing in organic solvents exposure and 1 control group) were examined using Ishihara-Panel, Lanthony Desaturated Panel D-15, Velhagen-Panel, and Tritan-Album. RESULTS: In the Velhagen-Panel 3% of the probands currently exposed to organic solvents, and 11% of formerly exposed probands developed a blue/yellow vision defect for the right eye. All control subjects perfectly finished this panel. In the Tritan-Album 3% of currently exposed subjects and 26% of formerly exposed painters expressed a blue/yellow vision defect for the right eye, but also 7% of controls showed anomalies. Similar results were found for both panels with the left eye. The CCI difference in the D-15 test was significant between all three groups. CONCLUSION: The impaired colour vision may also be an important indicator of neuro-ophthalmological effects after long-term occupational exposure to organic solvents.

The psychological effects of exposure to mixed organic solvents on car painters.

PURPOSE: Interest in diseases of the nervous system resulting from occupational exposure to mixed organic solvents has greatly increased. The aim of our study was to identify preclinical effects of low-level chronic solvent exposure on the central nervous system in car painters by assessing their cognitive performance. METHODS: This psychological study involved 169 clinically healthy male volunteers (84 car painters and 85 controls) and is part of a comprehensive study investigating effects of solvent exposure. The test battery included paper-pencil tests (vocabulary test, block design test, c.i., d2 test), computer-based tests (digit span test, simple-choice reaction time test), and a questionnaire to assess the participants' mental state. RESULTS: Car painters with long-term exposure to solvents showed psychological deviations such as deficits in concentration, memory and reaction time compared to unexposed subjects. The significant differences between the two groups were confirmed by multivariate statistical analysis. CONCLUSIONS: Our study displayed psychological effects associated with long-term solvent exposure in concentrations below German threshold limit values. These findings emphasize the necessity to promote the resolute compliance with occupational safety and health regulations in affected companies.

[Experiences with the psychometric system combitest in the evaluation of the postnarcotic arousal period]. / Erfahrungen beim Einsatz des Psychometriesystems COMBITEST bei der Untersuchung des Aufwachverhaltens im Anschluss an Allgemeinanästhesien.

Since the introduction of new German health legislation, operations formerly performed on an in-patient basis have been transferred to the out-patient sector. Due to this development evaluating the postnarcotic arousal period has become increasingly important. Vigilance, reactivity and state of health determine a patient's well-being and time of discharge after an operation and consequently influence economical and legal issues. Former studies investigating the arousal period have mainly focused on patient interviews and assessment of patients by staff. The aim of our study was to objectivize postnarcotic vigilance and reactivity (critical flicker fusion frequency and multiple choice reaction) using neurophysiological monitoring (psychometric system Combitest 2: CFF, EWR, MWR). The applied methods have been defined as valid, reliable and sensitive tools to investigate this question. Inter-individual and gender-specific differences as well as variations due to the type of anaesthetic employed can be detected. The study participants assessed the test battery's practicability and overall described this method as easy to use. Vigilance (CFF), basic and complex sensomotor reactivity (performance in simple and multiple choice tests) were restored after a postanaesthetic phase of 90 minutes (narcotics applied: isofluran and propofol). The results show that the use of modern anaesthetics does not justify the current medical and legal claim to 6 hours of postanaesthetic observation. The computer-supported psychometric system Combitest 2 has recently been used to assess the arousal phase after application of different types of anaesthetical procedures (TIVA, volatile, balanced) and has been found to be superior to parameters traditionally used to assess discharge criteria (time until patient opens eyes, orientation concerning time and location, time spent in postanaesthetic recovery room). Besides employing our study methods, subjective factors such as well-being and pain sensation should also be evaluated when determining the time of a patient's discharge.

[Neurotoxicity due to long-standing exposure to lead]. / Neirotoksicheskie éffekty mnogoletnei ékspozitsii svintsom.

Using computer psychometric tests characterizing central nervous system functions, the authors proved that workers contacting lead have altered visual perception time, rate and accuracy of motor functions, weaker short-term memory. Correlation analysis revealed no relationship between the changes obtained and the acting concentration. Research obviously should be parallel to studies of health state parameters.

Development of a pollution prevention and energy efficiency clearinghouse for biomedical research facilities.

This is the report of the National Association of Physicians for the Environment Committee on Development of a Pollution Prevention and Energy Efficiency Clearinghouse for Biomedical Research Facilities from the Leadership Conference on Biomedical Research and the Environment held at the National Institutes of Health in Bethesda, Maryland, on 1--2 November 1999. A major goal of the conference was the establishment of a World Wide Web-based clearinghouse, which would lend tremendous resources to the biomedical research community by providing access to a database of peer-reviewed articles and references dealing with a host of aspects of biomedical research relating to energy efficiency, pollution prevention, and waste reduction. A temporary website has been established with the assistance of the U.S. Environmental Protection Agency (EPA) Regions III and IV, where a pilot site provides access to the EPA's existing databases on these topics. A system of peer review for articles and promising techniques still must be developed, but a glimpse of topics and search engines is available for comment and review on the EPA Region IV-supported website (http://wrrc.p2pays.org/).

[Approaches to evaluation of occupational lead load in copper industry enterprise]. / Podkhody k otsenke professional'noi nagruzki svintsom na predptriiatii mednoi promyshlennosti.

Evaluation of copper industry in Saxony revealed serum lead concentrations in 109 workers under the allowable level accepted in Germany for workers contacting lead, ambient lead concentrations in air of workplace rarely 2-4 times over the allowable level. The authors recommend these parameters of exposure as a basis for evaluating health state of workers.