A large common-source outbreak of norovirus gastroenteritis in a hotel in Singapore, 2012.

An outbreak of gastroenteritis affected 453 attendees (attack rate 28·5%) of six separate events held at a hotel in Singapore. Active case detection, case-control studies, hygiene inspections and microbial analysis of food, environmental and stool samples were conducted to determine the aetiology of the outbreak and the modes of transmission. The only commonality was the food, crockery and cutlery provided and/or handled by the hotel's Chinese banquet kitchen. Stool specimens from 34 cases and 15 food handlers were positive for norovirus genogroup II. The putative index case was one of eight norovirus-positive food handlers who had worked while they were symptomatic. Several food samples and remnants tested positive for Escherichia coli or high faecal coliforms, aerobic plate counts and/or total coliforms, indicating poor food hygiene. This large common-source outbreak of norovirus gastroenteritis was caused by the consumption of contaminated food and/or contact with contaminated crockery or cutlery provided or handled by the hotel's Chinese banquet kitchen.

Characteristics of a widespread community cluster of H275Y oseltamivir-resistant A(H1N1)pdm09 influenza in Australia.

BACKGROUND: Oseltamivir resistance in A(H1N1)pdm09 influenza is rare, particularly in untreated community cases. Sustained community transmission has not previously been reported. METHODS: Influenza specimens from the Asia-Pacific region were collected through sentinel surveillance, hospital, and general practitioner networks. Clinical and epidemiological information was collected on patients infected with oseltamivir-resistant viruses. RESULTS: Twenty-nine (15%) of 191 A(H1N1)pdm09 viruses collected between May and September 2011 from Hunter New England (HNE), Australia, contained the H275Y neuraminidase substitution responsible for oseltamivir resistance. Only 1 patient had received oseltamivir before specimen collection. The resistant strains were genetically very closely related, suggesting the spread of a single variant. Ninety percent of cases lived within 50 kilometers. Three genetically similar oseltamivir-resistant variants were detected outside of HNE, including 1 strain from Perth, approximately 4000 kilometers away. Computational analysis predicted that neuraminidase substitutions V241I, N369K, and N386S in these viruses may offset the destabilizing effect of the H275Y substitution. CONCLUSIONS: This cluster represents the first widespread community transmission of H275Y oseltamivir-resistant A(H1N1)pdm09 influenza. These cases and data on potential permissive mutations suggest that currently circulating A(H1N1)pdm09 viruses retain viral fitness in the presence of the H275Y mutation and that widespread emergence of oseltamivir-resistant strains may now be more likely.

Increased detection in Australia and Singapore of a novel influenza A(H1N1)2009 variant with reduced oseltamivir and zanamivir sensitivity due to a S247N neuraminidase mutation.

A novel influenza A(H1N1)2009 variant with mildly reduced oseltamivir and zanamivir sensitivity has been detected in more than 10% of community specimens in Singapore and more than 30% of samples from northern Australia during the early months of 2011. The variant, which has also been detected in other regions of the Asia-Pacific, contains a S247N neuraminidase mutation. When combined with the H275Y mutation, as detected in an oseltamivir-treated patient, the dual S247N+H275Y mutant had extremely high oseltamivir resistance.

A new pandemic influenza A(H1N1) genetic variant predominated in the winter 2010 influenza season in Australia, New Zealand and Singapore.

Pandemic H1N1 influenza virus is of global health concern and is currently the predominant influenza virus subtype circulating in the southern hemisphere 2010 winter. The virus has changed little since it emerged in 2009, however, in this report we describe several genetically distinct changes in the pandemic H1N1 influenza virus. These variants were first detected in Singapore in early 2010 and have subsequently spread through Australia and New Zealand. At this stage, these signature changes in the haemagglutinin and neuraminidase proteins have not resulted in significant antigenic changes which might make the current vaccine less effective, but such adaptive mutations should be carefully monitored as the northern hemisphere approaches its winter influenza season.

Longitudinal sampling is required to maximize detection of intrahost A/H3N2 virus variants.

Seasonal human influenza viruses continually change antigenically to escape from neutralizing antibodies. It remains unclear how genetic variation in the intrahost virus population and selection at the level of individual hosts translates to the fast-paced evolution observed at the global level because emerging intrahost antigenic variants are rarely detected. We tracked intrahost variants in the hemagglutinin and neuraminidase surface proteins using longitudinally collected samples from 52 patients infected by A/H3N2 influenza virus, mostly young children, who received oseltamivir treatment. We identified emerging putative antigenic variants and oseltamivir-resistant variants, most of which remained detectable in samples collected at subsequent days, and identified variants that emerged intrahost immediately prior to increases in global rates. In contrast to most putative antigenic variants, oseltamivir-resistant variants rapidly increased to high frequencies in the virus population. Importantly, the majority of putative antigenic variants and oseltamivir-resistant variants were first detectable four or more days after onset of symptoms or start of treatment, respectively. Our observations demonstrate that de novo variants emerge, and may be positively selected, during the course of infection. Additionally, based on the 4-7 days post-treatment delay in emergence of oseltamivir-resistant variants in six out of the eight individuals with such variants, we find that limiting sample collection for routine surveillance and diagnostic testing to early timepoints after onset of symptoms can potentially preclude detection of emerging, positively selected variants.

Identification of a Lineage D Betacoronavirus in Cave Nectar Bats (Eonycteris spelaea) in Singapore and an Overview of Lineage D Reservoir Ecology in SE Asian Bats.

Coronaviruses are a diverse group of viruses that infect mammals and birds. Bats are reservoirs for several different coronaviruses in the Alphacoronavirus and Betacoronavirus genera. They also appear to be the natural reservoir for the ancestral viruses that generated the severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus outbreaks. Here, we detected coronavirus sequences in next-generation sequence data created from Eonycteris spelaea faeces and urine. We also screened by PCR urine samples, faecal samples and rectal swabs collected from six species of bats in Singapore between 2011 and 2014, all of which were negative. The phylogenetic analysis indicates this novel strain is most closely related to lineage D Betacoronaviruses detected in a diverse range of bat species. This is the second time that coronaviruses have been detected in cave nectar bats, but the first coronavirus sequence data generated from this species. Bat species from which this group of coronaviruses has been detected are widely distributed across SE Asia, South Asia and Southern China. They overlap geographically, often share roosting sites and have been witnessed to forage on the same plant. The addition of sequence data from this group of viruses will allow us to better understand coronavirus evolution and host specificity.

Increased plasma levels of pancreatic polypeptide and decreased plasma levels of motilin in encopretic children.

OBJECTIVE: Abnormalities of hormones affecting gastrointestinal motility have been found in "functional" disorders of the gastrointestinal system in adults. One such disorder of childhood, encopresis, is frequently associated with constipation, the treatment of which often eliminates the soiling. We hypothesized that hormones affecting gastrointestinal motility were different between encopretic patients and matched controls. METHODS: Ten encopretic patients were matched by age, race, and sex with controls who had no history of encopresis or constipation. After an overnight fast, each child consumed a meal of Ensure, the amount of which was based on body weight. Plasma levels of gastrin, pancreatic polypeptide, cholecystokinin, motilin, thyroxine, estrogen, and insulin were measured 20 and 5 minutes before the meal, and 5, 10, 15, 30, 45, 60, 90, 120, 150, and 180 minutes after the meal. RESULTS: Postprandial levels of pancreatic polypeptide remained consistently higher and peaked earlier (P < .05) for encopretic patients. The motilin response was lower (P < .03) for encopretic children than for controls. CONCLUSIONS: We conclude that pancreatic polypeptide and motilin responses to a meal are different in encopretic children than in children in the control group. These gastrointestinal hormone findings may in part explain and/or be the result of the severe constipation that frequently underlies the fecal soiling found in these patients. These findings also suggest the motility of the stomach and small intestine may be abnormal in encopresis.

The incidence of cognitive dysfunction in an encopretic population in children.

Cognitive testing was conducted in a clinical population of encopretic patients. Testing included the Weschler Intelligence Scale for Children-Revised (WISC-R), the Wide Range Achievement Test (WRAT) and the Bender Visual-Motor Gestalt Test (BVMGT). Although Full Scale, Verbal and Performance Intelligence Quotients on the WISC-R were not statistically different from general population means, subtests in Arithmetic (p less than 0.001), Digit Span (p less than 0.001) and Coding (p less than 0.05) were. There was also a high incidence of Verbal/Performance discrepancies and statistically significant differences on the WRAT in Spelling (p less than 0.01) and Arithmetic (p less than 0.01). Visual-motor integration was delayed two or more years in 43% of the patients on the BVMGT. These findings are consistent with the diagnosis of specific learning disabilities. An increased frequency of learning disabilities in an encopretic population may represent a subset of patients with a distinct syndrome in this disorder. These findings may point to the central nervous system (CNS) as the common point of pathology for the encopresis and learning disabilities, or reflect a common insult to both the CNS and gastrointestinal tract. Since histories in these patients did not reveal significant pregnancy or neonatal risk factors, this association could be related to genetic or environmental issues. Health professionals should be alert to the possibility of an increased incidence of learning disabilities in their encopretic patients. Further controlled studies are needed to determine if this finding is present in other settings, and if so, to determine the etiology.

Nutrition and cardiovascular disease.

The association between nutrition and coronary heart disease is mainly due to the effect of nutrients on serum lipids and lipoproteins. Cholesterol intake does not play a very important role for plasma cholesterol although there is a strong interindividual difference in response. The intake of saturated fatty acids strongly negatively affects plasma low-density lipoprotein cholesterol concentration while mono- and polyunsaturated fatty acids are generally regarded as beneficial. Omega-3 fatty acids mainly decrease triglyceride concentration while omega-6 polyunsaturated fatty acids mainly affect low-density lipoprotein cholesterol. Other nutrients which affect risk for coronary heart disease are dietary fiber, calcium, magnesium, iron, antioxidants as well as vitamins. Dietary fiber decrease intake of calories and fat, while iron and antioxidants play a role in oxidative modification of low-density lipoproteins. A low intake would lead to an accelerated uptake of low-density lipoprotein into the macrophage. Yet, intervention studies have not shown conclusively the benefit of a high-dose supplementation of the antioxidants vitamin E or beta-carotene on coronary heart disease. Homocysteine plasma concentration is influenced by folate as well as vitamin B6 and B12. Whether a high-dose supplementation with these substances does not only decrease plasma concentrations of homocysteine but also positively influence the course of coronary heart disease remains to be established.

Coaches' goals for young children in a recreational sports program.

Almost all children participate in sports at some time, and programs are being established for even younger children. Adults who coach the children largely determine what the children's sports experience will be. Coaches' perceptions of what is important for the young children they instruct have not yet been carefully investigated. This study was designed, therefore, to determine coaches' goals for young children. Data were gathered by use of an attitude questionnaire administered to 29 coaches of a recreational basketball program of children ranging in age from 6 to 10 years old. Of the 12 goals, feeling part of a team, learning to do my best, and having fun and excitement were most highly rated as extremely important, while becoming popular was lowest rated as not important. Results revealed that coaches in general are able to clearly define their goals and priorities, and these goals seem developmentally appropriate for the children. However, coaches make little differentiation in goals based on age.

Encopresis responsive to non-psychiatric interventions. With remittance of familial psychopathology.

A case report of an adolescent with primary encopresis is presented in which the patient responded to standard medical interventions despite apparent significant psychopathology in the child and family. Psychological symptoms proved to be secondary and resolved with successful medical treatment and with attending to other concerns, such as this patient's school problems. Behavioral and medical literature related to encopresis is reviewed. It indicates that encopresis is associated more consistently with physiological abnormalities of the bowel rather than definable psychological disorders.

Young children in recreational sports. Participation motivation.

More than 20 million children between ages 6 and 16 years participate in nonschool sports programs, with increasingly more programs being established for younger-age children. A questionnaire based on previous research with adolescent athletes was developed to determine participation motivation of 6- to 10-year-old children. Three hundred fourteen boys participating in a basketball program were interviewed privately with this instrument. Of the 12 participation motivation items, "learn to do my best," "learn and improve skills," "have a coach to look up to," and "get stronger and healthier" were most highly rated, whereas "win games" and "become popular" were rated least important. When responses of 6 and 7 year olds were compared with those of 9 and 10 year olds, older children rated "feel part of a team", "have fun and excitement" and "be with and make new friends" higher at a statistically significant level (p less than 0.05). Older children rated "win games" and "become popular" lower at a statistically significant level (p less than 0.05). The authors conclude that young children in this recreational sports program make significant distinctions in their participation motivation and that some aspects of participation that motivate them change as they grow older.

Application of pharmacokinetics in the diagnosis of chemical abuse in Münchausen syndrome by proxy.

The case of a preschooler who was chemically abused in Münchausen syndrome by proxy is presented. Though there were many clinical characteristics that raised suspicions of this syndrome, the definitive diagnosis was made by the application of pharmacokinetics during a hospital admission. This is the first reported use of this diagnostic approach to confirm this elusive disorder. Standard pharmacological equations can be used to provide this information from pharmacokinetic data which are readily available in most hospital settings.

Respiratory chain proteins.

Mammalian mitochondrial DNA codes for 13 proteins, which are all components of energy transducing enzyme complexes of the respiratory chain, i.e. the complexes which translocate protons across the inner mitochondrial membrane. The number of subunits of these enzyme complexes increase with increasing evolutionary stage of the organism. The additional nuclear coded subunits of the enzyme complexes from higher organisms are involved in the regulation of respiration, as demonstrated by the influence of intraliposomal ATP and ADP on the reconstituted cytochrome c oxidase (COX) from bovine heart. This regulation is not found with the reconstituted enzyme from P. denitrificans, which lacks the nuclear coded subunits. Some of the nuclear coded subunits occur in tissue-specific isoforms, as reported for COX and NADH dehydrogenase. Tissue-specific regulation of COX activity is also demonstrated by the differential effects of intraliposomal ADP on the kinetics of reconstituted COX from bovine liver and heart, which differ in subunits VIa, VIIa and VIII. At least 3 different COX isozymes occur in bovine liver, heart or skeletal muscle and smooth muscle. An evolutionary relationship between COX subunits VIa and VIc and between VIIa and VIIb is suggested based on the crossreactivity of monoclonal antibodies, amino acid sequence homology and hybridization at low stringency of PCR-amplified cDNAs for subunits VIa-1, VIa-h and VIc from the rat.

Genetic variability in progranulin contributes to risk for clinically diagnosed Alzheimer disease.

OBJECTIVE: Loss-of-function mutations in the progranulin gene (PGRN) were identified in frontotemporal lobar degeneration (FTLD) with ubiquitin-immunoreactive neuronal inclusions (FTLD-U). We assessed whether PGRN also contributes to genetic risk for Alzheimer disease (AD) in an extended Belgian AD patient group (n = 779, onset age 74.7 +/- 8.7 years). METHODS: A mutation analysis of the PGRN coding region was performed. The effect of missense mutations was assessed using in silico predictions and protein modeling. Risk effects of common genetic variants were estimated by logistic regression analysis and gene-based haplotype association analysis. RESULTS: We observed seven missense mutations in eight patients (1.3%). Convincing pathogenic evidence was obtained for two missense mutations, p.Cys139Arg and p.Pro451Leu, affecting PGRN protein folding and leading to loss of PGRN by degradation of the misfolded protein. In addition, we showed that PGRN haplotypes were associated with increased risk for AD. CONCLUSIONS: Our data support a role for PGRN in patients with clinically diagnosed Alzheimer disease (AD). Further, we hypothesize that at least some PGRN missense mutations might lead to loss of functional protein. Whether the underlying pathology in our cases proves to be AD, frontotemporal lobar degeneration, or a combination of the two must await further investigations.

Progranulin genetic variability contributes to amyotrophic lateral sclerosis.

OBJECTIVES: Null mutations in progranulin (PGRN) cause ubiquitin-positive frontotemporal dementia (FTD) linked to chromosome 17q21 (FTDU-17). Here we examined PGRN genetic variability in amyotrophic lateral sclerosis (ALS), a neurodegenerative motor neuron disease that overlaps with FTD at a clinical, pathologic, and epidemiologic level. METHODS: We sequenced all exons, exon-intron boundaries, and 5' and 3' regulatory regions of PGRN in a Belgian sample of 230 patients with ALS. The frequency of observed genetic variants was determined in 436 healthy control individuals. The contribution of eight frequent polymorphisms to ALS risk, onset age, and survival was assessed in an association study in the Belgian sample and a replication series of 308 Dutch patients with ALS and 345 Dutch controls. RESULTS: In patients with ALS we identified 11 mutations, 5 of which were predicted to affect PGRN protein sequence or levels (four missense mutations and one 5' regulatory variant). Moreover, common variants (rs9897526, rs34424835, and rs850713) and haplotypes were significantly associated with a reduction in age at onset and a shorter survival after onset of ALS in both the Belgian and the Dutch studies. CONCLUSION: PGRN acts as a modifier of the course of disease in patients with amyotrophic lateral sclerosis, through earlier onset and shorter survival.

[In vitro studies of the effect of different mixture proportions of omega-3 and omega-6 fatty acids on thrombocyte aggregation and thromboxane synthesis in human thrombocytes]. / In-vitro-Untersuchungen zum Einfluss verschiedener Mischungsverhältnisse an omega-3- und omega-6-Fettsäuren auf die Thrombozytenaggregation und Thromboxansynthese in Humanthrombozyten.

In order to estimate the influence of the tested fatty acids on platelet aggregation, synthesis of prostaglandin E and thromboxane B in vitro, platelet rich plasma (PRP) was incubated with the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), with linoleic acid as representative of the omega-6 fatty acids, as well as with mixtures of EPA and DHA and all fatty acids, resp., with and without addition of alpha-tocopherol. For the determinations, platelets were prepared from blood of young adult male volunteers (age 26.6 +/- 8 years). Platelet aggregation and synthesis of thromboxane were measured after 30 and 60 min of incubation. Smoking habits were not regarded. The incubation of platelets with DHA and EPA itself, as well as the mixture of fatty acids dominated by omega-3 fatty acids (omega-3/omega-6 = 15/1) caused a significant decrease (p less than 0.05) of collagen-induced platelet aggregation. Tocopherol, linoleic acid, and the linoleic-acid-rich mixtures (omega-3/omega-6 = 1/4) caused only a slight inhibition of platelet aggregation. No uniform influence of omega-3 fatty acids could be observed that showed their influence on synthesis of thromboxane to be of importance for the promotion of platelet aggregation. EPA and the mixture of EPA and DHA did decrease thromboxane synthesis significantly (p less than 0.05). On the other hand, single incubation with DHA as well as with linoleic acid rich mixtures caused a statistically not significant increase of rate of the synthesis, which did not increase the aggregation. This observation indicates the formation of less effective TXA3. An influence of tocopherol could also not be observed.

Influence of a single parenteral application of a 10% fish oil emulsion on plasma fatty acid pattern and the function of thrombocytes in young adult men.

50 ml of a 10% fish oil emulsion (41% omega-3 fatty acids of total fatty acids) were infused for 1 h into the arm vein of young, healthy, male volunteers. The fatty acid composition of the plasma, aggregation of the blood platelets as well as the thromboxane synthesis, were measured before the beginning of infusion, 20, 60, 120, 360 and 1,440 min after the start of the fat infusion. In the first 60 min, the fatty acid composition of the plasma changed in correspondence with the supplied fatty acid pattern. At the end of the investigation it was again within the normal range. As a result of fat application thromboxane synthesis was reduced and the aggregation of the platelets was inhibited but it was normalized by the 1,440-min value. Fish oil emulsions might be beneficial for parenterally fed patients with a high risk of thrombosis. Therefore the performance of further investigations using a varying dosage and multiple application can be recommended.

Design of multistable RNA molecules.

We show that the problem of designing RNA sequences that can fold into multiple stable secondary structures can be transformed into a combinatorial optimization problem that can be solved by means of simple heuristics. Hence it is feasible to design RNA switches with prescribed structural alternatives. We discuss the theoretical background and present an efficient tool that allows the design of various types of switches. We argue that both the general properties of the sequence structure map of RNA secondary structures and the ease with which our design tool finds bistable RNAs strongly indicates that RNA switches are easily accessible in evolution. Thus conformational switches are yet another function for which RNA can be employed.