Presence of human non-polio enterovirus and parechovirus genotypes in an Amsterdam hospital in 2007 to 2011 compared to national and international published surveillance data: a comprehensive review.

Enteroviruses (EV) and human parechoviruses (HPeV) are endemic worldwide. These infections are a constant cause of hospitalisation and severe disease, predominantly in young children and infants. Coordinated monitoring and surveillance are crucial to control these infections. We have monitored EV and HPeV epidemiology in Amsterdam from 2007 to 2011 with real-time RT-PCR and direct genotyping, facilitating highly sensitive surveillance. Moreover, we conducted a literature survey of existing surveillance data for comparison. Only 14 studies were identified. While HPeV1 was most frequently detected in Amsterdam, EV-B viruses dominated nationally and internationally. Furthermore, the top 10 strains detected differed yearly and per study. However, detection and typing methods were too varied to allow direct comparison and comprehension of the worldwide distribution and circulation patterns of the different genotypes. This limited a direct response to anticipate peaks. Uniform European monitoring programmes are essential to aid prediction of outbreaks and disease management.

Evaluation of two (semi-)nested VP1 based-PCRs for typing enteroviruses directly from cerebral spinal fluid samples.

Human enteroviruses (EVs) are the leading cause of CNS-associated disease in childhood. Identification of the EV types that patients are infected with is essential for monitoring outbreaks, the emergence of new types or variants, epidemiological surveillance and contributes to patient management. Rapid and sensitive molecular detection methods are frequently used to detect EVs/HPeVs directly from CSF. This requires that sensitive EV typing methods from CSF material need to be developed. In the present study two nested PCR-based typing assays were evaluated. The performance of the EV-A and -B specific nested PCR protocol and the Codehop-based PCR protocol were analyzed with several TCID(50)-titrated EV-A to D strains and 22 EV positive CSF samples. The EV-A and -B protocol was found to be more sensitive than the Codehop protocol. The Codehop protocol showed a high degree of aspecific amplification products when run on a gel, and required additional gel purification. The detection limit of the two protocols varied between the types, ranging from 0.1TCID(50)/mL sample to 10(6)TCID(50)/mL sample. From the 22 EV positive CSF samples, 15 (68%) samples were typed using either protocol. All samples were characterized as members of species B (E30 (9), CAV9 (2), E6 (1), E11 (1), E21 (1), E25 (1)). Three samples (E30 (2) and E25 (1)) could only be typed using the EV-B protocol. In this study, selected EV strains could be typed using both assays at low virus concentrations, typically found in CSF. However, the EV-A and -B protocol was more sensitive than the Codehop protocol for primary typing of CSF samples.

The effect of genetic polymorphism of cytochrome P450 CYP2C9 on phenytoin dose requirement.

The cytochrome P450 enzyme CYP2C9 catalyses the metabolism of numerous therapeutic agents, including the anti-epileptic drug phenytoin. CYP2C9 is genetically polymorphic: two allelic variants are known, CYP2C9*2 and CYP2C9*3, differing from the wild-type CYP2C9*1 by a single point mutation. Both mutant alleles are associated with markedly impaired metabolic capacity for many CYP2C9 substrates compared to the wild-type, resulting in raised serum drug levels upon a given dose. Because this may be relevant in treatment with phenytoin, we studied the effect of CYP2C9 genotype on phenytoin dose requirement in a group of 60 epileptic patients on long-term phenytoin therapy. CYP2C9 genotyping was performed by polymerase chain reaction analysis, phenytoin serum concentrations were measured by high-performance liquid chromatography analysis and related to the maintenance doses. For patients carrying at least one mutant CYP2C9 allele (n = 17), the mean phenytoin dose required to achieve a therapeutic serum concentration was about 37% lower than the mean dose required by wild-type individuals (199 mg/day versus 314 mg/day; P < 0.01). A low maintenance dose (< 200 mg/day) sufficed for 47% of carriers, while 58% of normals required a high dose (> 300 mg/day) for an effective serum level. The results show that there is a strong association between CYP2C9 allelic variants and phenytoin dose requirement. Since phenytoin has a narrow therapeutic index and genotyping may be carried out rapidly and at low cost, dosage adjustment based on CYP2C9 genotype, especially at the induction of therapy, would be of value in order to lower the risk of concentration dependent drug intoxications in carriers.

[Maintenance dose requirement for phenytoin is lowered in genetically impaired drug metabolism independent of concommitant use of other antiepileptics]. / Benodigde dagdosis fenytoïne verlaagd bij genetisch bepaald traag geneesmiddelmetabolisme en onbeïnvloed bij gelijktijdig gebruik van andere anti-epileptica.

OBJECTIVE: To investigate the effect of genetically determined impaired drug metabolism and of the use of comedication on phenytoin maintenance dose requirement. DESIGN: Descriptive. METHOD: In 60 patients on long-term phenytoin therapy the concentration of phenytoin in serum was measured and CYP2C9 genotyping was performed (mutant alleles of CYP2C9 are associated with impaired phenytoin metabolism). In addition, the use of other antiepileptics concurrently with phenytoin was reviewed: phenobarbital, carbamazepine and valproic acid. CYP2C9 genotype and comedication were connected to phenytoin daily dose requirement. The 60 patients were 38 men and 22 women, between 16 and 74 years of age, and all mentally disabled. RESULTS: Genotyping revealed that of the 60 patients 38% (n = 23) carried at least one mutant CYP2C9 allele. Their mean dose of phenytoin was 199 mg dd, while the mean required maintenance dose in non-carriers (n = 37) was 287 mg dd (p < 0.01). When the use of comedication was taken into account, it appeared that the mean phenytoin daily doses in the different comedication groups did not differ significantly. CONCLUSION: Patients with a genetically determined impaired phenytoin metabolism required on average a 30% lower dose than those with a normal metabolism, while the concomitant use of other antiepileptics appeared to have no effect on phenytoin dose requirement.

Retrospective study of doctors' "end of life decisions" in caring for mentally handicapped people in institutions in The Netherlands.

OBJECTIVES: To gain insight into the reasons behind and the prevalence of doctors' decisions at the end of life that might hasten a patient's death ("end of life decisions") in institutions caring for mentally handicapped people in the Netherlands, and to describe important aspects of the decisions making process. DESIGN: Survey of random sample of doctors caring for mentally handicapped people by means of self completed questionnaires and structured interviews. SUBJECTS: 89 of the 101 selected doctors completed the questionnaire. 67 doctors had taken an end of life decision and were interviewed about their most recent case. MAIN OUTCOME MEASURES: Prevalence of end of life decisions; types of decisions; characteristics of patients; reasons why the decision was taken; and the decision making process. RESULTS: The 89 doctors reported 222 deaths for 1995. An end of life decision was taken in 97 cases (44%); in 75 the decision was to withdraw or withhold treatment, and in 22 it was to relieve pain or symptoms with opiates in dosages that may have shortened life. In the 67 most recent cases with an end of life decision the patients were mostly incompetent (63) and under 65 years old (51). Only two patients explicitly asked to die, but in 23 cases there had been some communication with the patient. In 60 cases the doctors discussed the decision with nursing staff and in 46 with a colleague. CONCLUSIONS: End of life decisions are an important aspect of the institutionalised care of mentally handicapped people. The proportion of such decisions in the total number of deaths is similar to that in other specialties. However, the discussion of such decisions is less open in the care of mental handicap than in other specialties. Because of distinctive features of care in this specialty an open debate about end of life decisions should not be postponed.

Overexpression of human insulin-like growth factor-II in transgenic mice causes increased growth of the thymus.

In order to determine the effects of IGF-II overexpression on growth of mice, transgenic mice were produced carrying one of three different H-2Kb human IGF-II minigenes in which different non-coding exons (exon 5, truncated exon 5 or exon 6) preceded the coding exons 7, 8 and 9. These were spaced by truncated introns and for proper polyadenylation an SV40 polyadenylation signal was incorporated. The highest levels of IGF-II minigene mRNA expression were found in lines containing the truncated exon 5 construct (II5'). Those containing exon 6 (II6) had less expression and 5 constructs (II5) gave only moderate levels of mRNA expression. In general mRNA expression was highest in thymus and spleen, low in liver and kidney and absent in the brain. In addition, one II5' line showed expression in the brain. Serum IGF-II levels at 8 weeks of age were increased 7- to 8-fold in homozygous transgenic lines with construct II5' without brain expression and 2- to 3-fold in the one that showed expression in the brain; serum IGF-I levels were unchanged. Serum IGFs in the lines containing the constructs II5 and II6 were not different from those of the controls. In all cases body length and weight as well as the weight of several organs such as brain, liver, kidneys, heart and spleen when expressed as a function of age did not differ from controls. Only the thymus showed a significant increase in weight in the transgenics II5'. Inbreeding of 2 lines containing construct II5' with pituitary deficient Snell dwarf mice did not influence body length or weight despite increased serum IGF-II levels. Again the thymus showed a marked increase in growth. The biological activity of the IGF-II peptide was further demonstrated by increased serum IGF-binding protein-3 in the transgenic dwarf mice, as shown by Western ligand blotting. In summary, overexpression of IGF-II in transgenic normal and dwarf mice does not affect overall body growth, but causes increased growth of the thymus. This suggests a role for IGF-II in thymic development by paracrine/autocrine action.

Changes in zinc metabolism after burns: observations, explanations, clinical implications.

Zinc in plasma and urine and serum albumin and alpha 2-macroglobulin were measured in 48 patients with burns. Mean total burned surface area amounted to 18%, ranging from 2 to 55%, and mean hospitalization time amounted to 35 days, ranging from 10 to 124 days. All parameters showed a decrease during the first two post-burn days. Minimal values were reached on days 2 and 3 for plasma and urine zinc, and between days 5 and 10 for the proteins. Thereafter, values increased, rapidly for both plasma and urinary zinc, more slowly for albumin and alpha 2-macroglobulin. The ratio R of the total plasma zinc minus the alpha 2-macroglobulin concentration to the albumin concentration is postulated as an indicator for zinc deficiency. From values of R and of the urinary zinc excretion, conclusions can be drawn about various processes of the zinc metabolism that may occur during the acute stage following the thermal accident and during the stages of tissue demarcation and of recovery. These processes are discussed in terms of possible temporary and/or local zinc deficiency. Evidence is presented that zinc administration in only indicated during the final stages of recovery in case of inadequate dietary intake.

Urinary zinc excretion in a patient with burns: a caveat when using bladder catheters in urine zinc studies.

The results of a longitudinal study on plasma and urine zinc concentrations in a patient with 68 per cent burns are described. Special attention was paid to possible zinc contamination due to the use of bladder catheters for urine sampling. The course of the plasma and urine zinc concentrations during the first 50 days in hospital was comparable to those reported in the literature. It was shown that surgery was associated with a pronounced rise in urine zinc level, while catheter replacement caused no significant increase in urinary zinc content in this particular patient. However, measurements of zinc content of and zinc release from three types of bladder catheters showed that the two types of latex-based catheters contained about 0.25 per cent zinc, versus 0.01 per cent in the siliconelastomer equivalent. During an 8 h perfusion latex catheters released about 1.2 mumols zinc (2.5 mumols/l). Possible errors in urine zinc determinations due to zinc contamination from catheters are calculated. Recommendations are made for minimizing these errors.

Hexose monophosphate shunt activity in erythrocytes related to cell age.

Erythrocytes were separated by age using a combination of density centrifugation and counterflow centrifugation and tested for basal activity of the hexose monophosphate shunt (HMP-shunt) as well as the methylene blue-stimulated maximal capacity by measuring CO2 production. No significant differences were found in basal HMP-shunt activity, but the maximal methylene blue-stimulated activity of old erythrocytes reached only half of the activity of the total cell population. The maximal HMP-shunt activity showed a significant correlation with hexokinase activity, but not with glucose-6-phosphate dehydrogenase activity in all but the youngest cells. The sensitivity to oxidative stress was tested by measuring the kinetics of pyruvate kinase isolated from erythrocytes incubated in presence and absence of methylene blue. Pyruvate kinase kinetics were affected more in the old cell population than in the total cell population: the K0.5 for phosphoenol-pyruvate increased four times in the unseparated cells and eight times in old cells.