Validation of a KHV antibody enzyme-linked immunosorbent assay (ELISA).

Koi herpesvirus (KHV) causes KHV disease (KHVD). The virus is highly contagious in carp or koi and can induce a high mortality. Latency and, in some cases, a lack of signs presents a challenge for virus detection. Appropriate immunological detection methods for anti-KHV antibodies have not yet been fully validated for KHV. Therefore, it was developed and validated an enzyme-linked immunosorbent assay (ELISA) to detect KHV antibodies. The assay was optimized with respect to plates, buffers, antigens and assay conditions. It demonstrated high diagnostic and analytical sensitivity and specificity and was particularly useful at the pond or farm levels. Considering the scale of the carp and koi industry worldwide, this assay represents an important practical tool for the indirect detection of KHV, also in the absence of clinical signs.

Isolation and partial characterization of a novel virus from different carp species suffering gill necrosis - ultrastructure and morphogenesis.

Two isolates of a novel enveloped RNA virus were obtained from carp and koi carp with gill necrosis. Both isolates behaved identically and could be propagated in different cyprinid cell lines forming large syncytia. The virus was sensitive to lipid solvents and neither exhibited haemadsorption/haemagglutination nor reverse transcriptase activity. Mature virus particles displayed a spherical shape with diameter of 100-350 nm after negative staining and 100-300 nm in ultrathin sections, covered by short projections of 8-10 nm in length. Maturation of virus progeny was shown to occur by budding and envelopment of the filamentous helical nucleocapsids at the cell surface. A detailed comparison of ultrastructure and morphogenesis of the novel virus isolates with selected arena-, ortho- and paramyxoviruses as possible candidates for evaluation of taxonomic classification yielded no consistency in all phenotypic features. Thus, on the basis of ultrastructure the novel virus isolates could not be assigned unequivocally to any established virus family.

Impact of propofol on mid-latency auditory-evoked potentials in children.

BACKGROUND: Propofol is increasingly used in paediatric anaesthesia, but can be challenging to titrate accurately in this group. Mid-latency auditory-evoked potentials (MLAEPs) can be used to help titrate propofol. However, the effects of propofol on MLAEP in children are unclear. Therefore, we investigated the relationship between propofol and MLAEP in children undergoing anaesthesia. METHODS: Fourteen healthy children aged 4-16 yr received anaesthesia for elective surgery. Before surgery, propofol was administered in three concentrations (3, 6, 9 µg ml(-1)) through a target-controlled infusion pump using Kataria and colleagues' model. MLAEPs were recorded 5 min after having reached each target propofol concentration at each respective concentration. Additionally, venous propofol blood concentrations were assayed at each measuring time point. RESULTS: Propofol increased all four MLAEP peak latencies (peaks Na, Pa, Nb, P1) in a dose-dependent manner. In addition, the differences in amplitudes were significantly smaller with increasing propofol target concentrations. The measured propofol plasma concentrations correlated positively with the latencies of the peaks Na, Pa, and Nb. CONCLUSIONS: Propofol affects MLAEP latencies and amplitudes in children in a dose-dependent manner. MLAEP measurement might therefore be a useful tool for monitoring depth of propofol anaesthesia in children.

An unreported complication of intravenously administered ibuprofen: gastrointestinal bleeding.

Ibuprofen is used for the closure of ductus arteriosus either intravenously or enterally. Although intraventricular hemorrhage, necrotizing enterocolitis, bronchopulmonary dysplasia, transient renal failure, oliguria, hyponatremia and thrombocytopenia are reported complications during or after ibuprofen treatment, gastrointestinal bleeding, to our knowledge, has not been reported previously. We herein report a premature newborn, in whom ibuprofen was used intravenously for the closure of ductus arteriosus and gastrointestinal bleeding developed as a complication, and aim to discuss this rare adverse effect. In conclusion, we emphasize the importance of close follow-up of premature newborns during intravenous ibuprofen treatment considering also the other rare systemic side effects reported in the literature.

Effects of increasing sevoflurane MAC levels on mid-latency auditory evoked potentials in infants, schoolchildren, and the elderly.

BACKGROUND: Detection of mid-latency auditory evoked potentials (MLAEPs) is a technology to monitor central nervous structures. As seen in adults and children, general anaesthesia influences the MLAEP latencies. MLAEP detection seems to be a promising tool to assess different levels of anaesthesia depth in adults and children. METHODS: MLAEPs were recorded in 10 infants (2 months-3 yr), 12 schoolchildren (6-14 yr), and 10 elderly (75-89 yr) under general anaesthesia with increasing concentrations of sevoflurane at steady state. In addition, MLAEPs were detected before and after the application of sufentanil. RESULTS: At all different ages, MLAEP latencies increased significantly with higher volume percentages of sevoflurane. These results were also detectable when MAC values of sevoflurane were compared with MLAEP peaks. An age-dependent effect could be displayed as elderly people need lower absolute sevoflurane concentrations to achieve the same MLAEP peak increase. Overall, the application of sufentanil under steady-state sevoflurane application at 1 MAC did not importantly affect the MLAEP latencies. CONCLUSIONS: MLAEP latencies increase at the influence of sevoflurane in a dose-dependent manner and in relation to age. These results imply that MLAEP detection is a reasonable tool for monitoring hypnotic effects at all ages. Further studies are required to standardize MLAEP alterations related to effects of medication used for general anaesthesia at all different ages.

Susceptibility of koi x crucian carp and koi x goldfish hybrids to koi herpesvirus (KHV) and the development of KHV disease (KHVD).

Hybrids of koi, Cyprinus carpio x crucian carp, Carassius carassius and koi x goldfish, Carassius auratus, proved to be susceptible to koi herpesvirus (KHV, syn. CyHV-3) and developed KHV disease (KHVD). While hybrids of koi x goldfish were partly resistant to mortality following infection by immersion, most koi x crucian carp hybrids died after bath infection. KHV DNA was detected in dead fish but also in all surviving animals by different polymerase chain reactions (PCRs). According to these results, hybrid crossbreeding does not seem to prevent severe losses associated with KHV in terms of inducing KHVD. The present study showed severe losses after a waterborne KHV infection of between 35% and 100% in koi x goldfish and koi x crucian carp hybrids as well as in SPF carp.

Importance of plasma N-terminal pro B-type natriuretic peptide, epicardial adipose tissue, and carotid intima-media thicknesses in asymptomatic obese children.

This study aimed to analyze the variations of N-terminal pro B-type natriuretic peptide, epicardial adipose tissue thickness, and carotid intima-media thickness in childhood obesity. The study participants consisted of 50 obese children in the study group and 20 nonobese children referred for evaluation of murmurs who proved to have an innocent murmur and were used as control subjects. All the subjects underwent transthoracic echocardiographic examination for determination of left ventricular systolic function and mass index, myocardial tissue rates, and myocardial performance index. Epicardial adipose tissue thickness and carotid intima-media thickness also were measured during echocardiography. Serum N-terminal pro B-type natriuretic peptide levels were measured at the time of evaluation. The left ventricle mass index was 40.21 + or - 10.42 g/m(2) in the obese group and 34.44 + or - 4.51 g/m(2) in the control group (p > 0.05). The serum N-terminal pro B-type natriuretic peptide level was 109.25 + or - 48.53 pg/ml in the study group and 51.96 + or - 22.36 pg/ml and in the control group (p = 0.001). The epicardial adipose tissue thickness was 5.57 + or - 1.45 mm in the study group and 2.98 + or - 0.41 mm in the control group (p = 0.001), and the respective carotid intima-media thicknesses were 0.079 + or - 0.019 cm and 0.049 + or - 0.012 cm (p = 0.001). The left ventricular systolic and diastolic functions showed no statistically significant correlations with N-terminal pro B-type natriuretic peptide levels, carotid intima-media thickness, or epicardial adipose tissue thickness values. The results show that measurement of serum N-terminal pro B-type natriuretic peptide level, carotid intima-media thickness, and epicardial adipose tissue thickness in asymptomatic obese children is not needed.

Highly sensitive fetal goat tongue cell line for detection and isolation of foot-and-mouth disease virus.

A fetal goat cell line (ZZ-R 127) supplied by the Collection of Cell Lines in Veterinary Medicine of the Friedrich Loeffler Institute was examined for susceptibility to infection by foot-and-mouth disease (FMD) virus (FMDV) and by two other viruses causing clinically indistinguishable vesicular conditions, namely, the viruses of swine vesicular disease and vesicular stomatitis. Primary bovine thyroid (BTY) cells are generally the most sensitive cell culture system for FMDV detection but are problematic to produce, particularly for laboratories that infrequently perform FMD diagnostic tests and for those in countries where FMD is endemic that face problems in sourcing thyroid glands from FMD-negative calves. Strains representing all seven serotypes of FMDV could be isolated in ZZ-R 127 cells with a sensitivity that was considerably higher than that of established cell lines and within 0.5 log of that for BTY cells. The ZZ-R 127 cell line was found to be a sensitive, rapid, and convenient tool for the isolation of FMDV and a useful alternative to BTY cells for FMD diagnosis.

Threshold value of subepicardial adipose tissue to detect insulin resistance in obese children.

AIM: Until now, the association between subepicardial adipose tissue (SAT), insulin resistance and intima-media thickness (IMT) has not been evaluated in obese children. In this study, we evaluated whether echocardiographic SAT is related to insulin resistance and IMT in obese children. SUBJECTS AND METHODS: A total of 46 obese subjects (10.2+/-2.5 years of age, 25 male patients) and 30 age- and gender-matched lean subjects (10.8+/-3.1 years of age, 13 male patients) were included in this study. The criterion for diagnosing obesity was defined as the body mass index (BMI) being over 97% percentile of the same gender and age. Serum triglyceride (TG), low- and high-density lipoprotein, cholesterol, glucose and insulin levels were measured during the fasting state. Each subject underwent a transthoracic echocardiogram and the SAT thickness was measured during end-diastole from the parasternal long-axis views. RESULTS: The obese subjects had significantly higher SAT thickness and IMT values compared with the subjects in the control group (5.7+/-1.4 vs 3.0+/-0.7 mm, 0.78+/-0.15 vs 0.51+/-0.11 mm, P=0.001, respectively). Simple linear regression analysis showed no significant correlation between SAT and insulin resistance (r=0.170, P=0.253), whereas there was significant correlation between SAT and BMI, age and IMT (r=0.625, P=0.02, r=0.589, P=0.001, r=0.343, P=0.02, respectively). As an optimal cutoff point, a SAT thickness of 4.1 mm determined insulin resistance with 90% sensitivity and 61% specificity. CONCLUSIONS: Our study showed that SAT was significantly correlated with age, BMI and IMT, but not insulin resistance. However, our findings suggest that a 4.1 mm cutoff of SAT thickness might be used as a simple, inexpensive and non-invasive screening method because of its ability to predict insulin resistance with high sensitivity in obese children.

Neutrophil hypersegmentation in children receiving angiotensin converting enzyme inhibitors.

Captopril and enalapril are the most commonly used angiotensin converting enzyme inhibitors in several cardiac diseases in children. On the other hand, the intrinsic renin-angiotensin system in the bone marrow might affect the growth of hematopoietic colonies and cellular production, proliferation and differentiation in physiological and pathological states. Starting with the hypothesis that inhibition of the renin-angiotensin system may have some effects on the hematopoietic system, including morphological changes within the granulocytes, we thus aimed to investigate prospectively whether the use of angiotensin converting enzyme inhibitors has any effect on the morphology, and especially segmentation, of neutrophils in peripheral blood. A total of 40 children with various heart diseases receiving either of two angiotensin converting enzyme inhibitors (captopril or enalapril) aged between 2 to 16 years were enrolled, and 40 healthy age- and sex-matched children were enrolled as controls. Complete blood count, peripheral blood smear, liver and renal function tests, and measurement of serum alkaline phosphatase, ferritin, vitamin B12 and folate levels were performed in all cases. Peripheral blood smears were viewed by two pediatric hematologists in a blinded manner. Neutrophil hypersegmentation was described as presence of five or more neutrophils with five well-separated lobes or at least one neutrophil with six or more lobes among 100 segmented neutrophils. The number of patients with neutrophil hypersegmentation in the study group was significantly higher than in the control group, and the mean lobe count in the study group was significantly higher than in the control group. Neutrophil hypersegmentation, as detected in patients using angiotensin converting enzyme inhibitors in the present study, has not been reported previously. Further studies aiming to explain the pathophysiological mechanism(s) underlying neutrophil hypersegmentation in patients receiving angiotensin converting enzyme inhibitors are needed.

Detection of European porcine reproductive and respiratory syndrome virus in porcine alveolar macrophages by two-colour immunofluorescence and in-situ hybridization-immunohistochemistry double labelling.

Two groups of five pigs aged 6 weeks were each infected oronasally with one of two different European isolates of porcine reproductive and respiratory syndrome virus (PRRSV). The animals were killed sequentially at 4, 7, 14 or 21 days post-inoculation for examination. The methods used consisted of histopathology, and mono- and double-labelling techniques based on in-situ hybridization, immunofluorescence and immunohistochemistry. Porcine alveolar macrophages (PAMs) contained large amounts of PRRSV antigen and PRRSV RNA, as shown by double labelling with (1) either PRRSV immunofluorescence or PRRSV-specific in-situ hybridization with digoxigenin-labelled riboprobes, and (2) immunolabelling with Mac 387 antibody for calprotectin. Expression of PRRSV-RNA was not detectable in cytokeratin-positive hypertrophic and proliferating pneumocytes or in cells of alveolar ducts or bronchiolar epithelium. The use of two-colour immunofluorescence with confocal laser scanning microscopy and double labelling with in-situ hybridization-immunohistochemistry showed that PAMs were the only pulmonary target cells. This contradicts earlier reports that epithelial pulmonary cells may also be infected by PRRSV.

Bruce treadmill test in healthy Turkish children: endurance time, heart rate, blood pressure and electrocardiographic changes.

Heart rate, systolic and diastolic blood pressure and electrocardiographic changes were studied in 80 healthy children during treadmill exercise with Bruce protocol. Mean duration of exercise in boys increased from 10.01 +/- 1.61 minutes at age four to six years with a mean body surface area of 0.75 +/- 0.06 m2, to 17.86 +/- 2.66 minutes at age 13 to 15 years with a mean body surface area of 1.48 +/- 0.10 m2. Mean endurance time in girls increased from 11.06 +/- 1.92 minutes at age four to six years with a mean body surface area of 0.76 +/- 0.08 m2, to 15.69 +/- 2.23 minutes at age 13 to 15 years with a mean body surface area of 1.41 +/- 0.07 m2. Mean maximal heart rate was 193.38 +/- 10.89 beats/minute in boys and 196.78 +/- 10.99 beats/minute in girls. The maximum level of systolic blood pressure attained at peak exercise was lower in girls. Although data of healthy Turkish children including mean endurance time, heart rate and blood pressure responses to exercise were consistent with the results from several countries using the Bruce protocol, mean maximal heart rates for all groups were slightly higher than those obtained with the Balke protocol. The data obtained from this study offers age- and size-appropriate normal data in both sexes in healthy Turkish children and may be used as reference values during treadmill exercise.

Short- and intermediate-term efficacy of amiodarone in infants and children with cardiac arrhythmia.

The antiarrhythmic effect of amiodarone was examined in this retrospective study in a group of 20 patients with a mean age of 8.5 +/- 6.7 years (range 42 days to 20 years, median 9 years). Five patients with atrial flutter, one patient with atrial fibrillation, two patients with an intermediate rhythm between atrial flutter and atrial fibrillation, four patients with chaotic atrial tachycardia, three patients with atrioventricular reentry tachycardia, two patients with junctional ectopic tachycardia, and three patients with ventricular arrhythmias were treated with amiodarone. The mean duration of therapy was 9.1 +/- 12.3 months (range 1 month to 4 years). Before amiodarone treatment, 18 patients had been unresponsive to various antiarrhythmic drugs (range 1-8, median 2). Two patients received amiodarone as an initial therapy. It was administered orally at a dose of 10 mg/kg once per day for 10 days and then decreased to 5 mg/kg once per day. Amiodarone was effective in 16 patients (80%). Side effects occurred in three patients, including thyroid dysfunction, elevation of liver enzymes, and keratopathy. All side effects disappeared upon cessation of the therapy. We recommend amiodarone for the treatment of childhood arrhythmias, especially for the refractory types.

Vasovagal syncope: asystole provoked by head-up tilt testing under sertraline therapy.

Syncope is defined as a sudden transient loss of consciousness. Vasovagally mediated hypotension and bradycardia are believed common, yet difficult to diagnose, causes of syncope in healthy children and adolescents. These episodes are often both sudden and sporadic in nature and, if recurrent and severe (malignant vasovagal syncope), can be a source of morbidity and possibly mortality. Head-up tilt testing has emerged as a useful investigation in patients who are thought to have recurrent vasovagal syncope with systemic hypotension, bradycardia, or both, and it has been suggested as a potential method to test for vasovagal episodes. Sertraline hydrochloride, a serotonin reuptake inhibitor, has been reported to be effective in preventing the vasovagal syncopal episodes in children and adults. Here, two cases of recurrent, unexplained syncope are presented. Both were under sertraline therapy and underwent provocative head-up tilt testing that resulted in asystole.

Interruption of the distal left pulmonary artery with pulmonary arteriovenous fistulas and atrial septal defect.

A seven-year-old boy with interrupted left pulmonary artery, arteriovenous fistulas and atrial septal defect is presented. The anomalies were identified by echocardiography and angiography. On pulmonary artery angiograms, left pulmonary artery discontinuity (as though the distal segment had been interrupted), diffuse granulary opacification of right lung fields, and early visibility of the left atrium were observed.

Detection of early anthracycline-induced cardiotoxicity in childhood cancer with dobutamine stress echocardiography.

Asymptomatic long-term survivors of childhood cancer treated with anthracyclines may have latent cardiac dysfunction which is undetected by commonly used echocardiographic methods. A more sensitive echocardiographic screening test, dobutamine stress echocardiography, was performed on 22 patients (mean age 9.10 +/- 3.79 years) treated with 75 to 450 mg/m2 of anthracyclines (mean 210.45 +/- 127.34) and results were compared with 22 healthy age-matched control subjects. Echocardiographic Doppler studies were performed after each dobutamine infusion of 0.5, 2.5, 5 and 10 micrograms/kg/min. Although left ventricular mass was decreased and end-systolic walls stress increased in the patient group when compared with the control subjects (p < 0.01 and p < 0.05, respectively), no differences were found between shortening fraction and ejection force in control subjects and patients, at rest and during each dobutamine infusion. A decreased mitral E/A ratio (ratio of early-to-late peak filling velocity) was demonstrated in anthracycline-treated patients only during dobutamine infusion (p < 0.01). Our data showed left ventricular diastolic dysfunction during intropic stimulation with dobutamine, and suggest that dobutamine stress echocardiography is a useful technique for evaluating the cardiac status of anthracycline-treated patients on a long-term basis.

Ambulatory electrocardiographic monitoring in healthy newborn infants.

Twenty-four hour ambulatory electrocardiographic monitoring is a useful method to document dysrhythmias and to assess treatment response. Various studies have been done in the pediatric age-group to determine normal heart rate values. In this study we determined the heart rate and rhythm patterns of 25 healthy newborn infants whose ages ranged between 3-10 days (mean 6.5 days). There were 15 males and 10 females. The maximum heart rate in these infants was 175-231 beats/min (207 +/- 14), minimum heart rate 60-121 beats/min (93 +/- 16) and the average heart rate was 130-161 beats/min (143 +/- 9). Five infants (20%) demonstrated marked sinus dysrhythmia, seven infants (28%) had ventricular premature contractions, two infants (8%) had supraventricular premature contractions, and five infants (20%) showed junctional rhythm disturbance. The sinus pause did not exceed 1.2 sec and there was no evidence of atrioventricular conduction disorders, or supraventricular-ventricular tachycardia attacks. Our results were consistent with previous studies carried out in newborn infants. Dysrhythmias were detected during 24-hour ambulatory electrocardiographic monitoring in our study group. Since they were generally benign, they need no treatment.

Variable clinical expression of Holt-Oram syndrome in three generations.

Holt-Oram syndrome is a distinct autosomal dominant entity presenting with upper limb defects and cardiac abnormality. No correlation between the severity of the heart and the limb defects has been established. Here we report variable clinical expression of Holt-Oram syndrome in three generations. The grandfather presented with typical upper limb defects: phocomelia of arms with three digits on each hand, congenital heart defect and narrow shoulders. His son manifested cardiac conduction disturbance with no congenital heart or skeletal defect. The granddaughter showed ventricular septal defect and moderate radial deviations of both hands with no obvious hypoplasia of the extremities. Clinical data of the presented family suggests lack of penetrance with respect to skeletal and structural cardiac abnormalities in the Holt-Oram syndrome.

Atrioventricular nodal reentrant tachycardia ablated from left atrial septum: clinical and electrophysiological characteristics and long-term follow-up results as compared to conventional right-sided ablation.

Radiofrequency catheter ablation or modification of the slow pathway is almost always performed on the right atrial side of the interatrial septum, however, this is not possible in rare cases. We evaluated the clinical and electrophysiological characteristics and long-term follow-up results of patients whose AVNRT could only be ablated from the left posterior atrial septum after repeated unsuccessful attempts on the right atrial side and to observe if they differ from those undergoing ablation with the conventional right-sided approach. Of 587 cases with symptomatic typical AVNRT, 9 patients (1.5%) in whom RF energy delivered to the right atrial septum with the integrated approach failed to ablate or modify the slow pathway were enrolled in the study group (group 1) while the others served as controls (group 2). There was no significant difference between the groups regarding clinical characteristics, dual AV nodal physiology, sinus cycle lengths, AH and HV intervals, procedural complication rates, or recurrence rates in the mean follow-up duration of 34 +/- 11 months. Only tachycardia cycle length (TCL) was significantly higher in group 1 than in group 2, which was mainly due to the difference in AH intervals (P < 0.001 for both). Slow pathway ablation was performed at the posteroseptal aspect of the mitral annulus in 6 and the midseptal aspect in 2 cases. In 1 case, attempts at ablation on the left atrial septum also failed. When the conventional right-sided approach fails to ablate or modify the slow pathway conduction, left-sided ablation can safely and effectively be employed, with success rates and long-term follow-up results comparable to the conventional right-sided approach.