Detection of Norwalk Virus and Other Genogroup 1 Human Caliciviruses by a Monoclonal Antibody, RecombinantAntigen-Based Immunoglobulin M Capture Enzyme Immunoassay

Sera obtained from two groups of adult volunteers infected with Norwalk virus (NV) and two groups of patients involved in two natural outbreaks were tested for NV-reactive immunoglobulin M (IgM) by use of a monoclonal antibody, recombinant-antigen-based IgM capture enzyme immunoassay (EIA). No NV-reactive IgM was detected in the preinoculation sera of 15 volunteers, and 14 of 15 showed NV-reactive antibodies postinfection with NV. All of the volunteers showed IgG seroconversion to NV. In the outbreak studies, all 9 persons in one outbreak and 19 of 24 in another outbreak had NV-reactive IgM. In the first outbreak, only three of nine seroconverted to NV, which was likely due to late collection of acute-phase sera. In the second outbreak, 21 of 24 showed IgG seroconversion to NV. Sequencing of viruses isolated from five stool samples selected from those in the second outbreak showed that they were human calicivirus (HuCV) genogroup 1 viruses related, but not identical, to NV. In the volunteer studies, NV-reactive IgM was first detected 8 days postinoculation. The time of development of NV-reactive IgM antibodies in natural outbreaks was estimated to be similar to that found in the volunteer studies. Sera from three Hawaii virus-infected volunteers, four Snow Mountain virus patients, and 80 healthy individuals were negative for NV-reactive IgM, indicating test specificity for HuCV genogroup I infections. This capture IgM EIA is suitable for diagnosis of NV and other HuCV genogroup I infections and is especially useful when sera and fecal samples have not been collected early in the course of an outbreak.     

Age-related alterations in dopamine and norepinephrine activity within microdissected brain regions of ovariectomized long evans rats

The ability of several stimuli which augment central catecholamine (CA) neuronal activity to reinitiate estrous cycles in old constant estrous (CE) rats suggests CA neuronal function is impaired with advanced age. We examined the effects of age on dopamine (DA) and norepinephrine (NE) levels and turnover rates within microdissected brain regions of previously normally cycling young (3-4 months old) and middle-aged (10 months old) and CE old (20-22 months old) Long Evans 2 weeks after ovariectomy. Steady-state DA concentrations were significantly decreased in old compared to young rats in the nucleus accumbens (34%), anterior hypothalamic nucleus (54%, NHA ), neurointermediate pituitary lobe (51%, NIL) and median eminence (74%, ME). The rate constant of DA loss, an estimate of neuronal activity, decreased in old versus young rats only in the preoptic area suprachiasmatica (60%, POAs ) and NHA (60%) and was unchanged or augmented in the 7 other regions. In contrast, a decline in DA turnover rate of 29-67% was observed in 6 of 9 regions in middle-aged rats and 45-81% in 5 of 9 regions in old rats. Steady-state NE concentrations similarly were significantly decreased in old versus young rats in the POAs (54%), medial forebrain bundle (44%), nucleus suprachiasmatica (49%) and ME (59%). The rate constant of NE loss progressively decreased with increasing age only in the POAs and was unchanged or augmented in other regions. Turnover rate of NE was decreased from 21 to 98% in 4 of 8 regions from old animals. A strong positive correlation was noted between the rate constant of NE (but not DA) loss measured in young rats and the magnitude of the age-related depletion in NE concentrations within specific brain regions. Collectively these data indicate that with increasing age: CA neuronal function is differentially altered in nuclei located along the preoptico-tuberal pathway; substantial declines in both DA and NE concentrations are the primary contributor to the reduced amine turnover noted in several of these regions; and the observed age-related alterations in CA turnover may contribute to impaired LH response and the persistent hyperprolactinemia in old CE rats.     

Flecainide: electrophysiologic and antiarrhythmic properties in refractory ventricular tachycardia

Twenty-two patients with coronary artery disease and spontaneous ventricular tachycardia (VT) or ventricular fibrillation (VF) underwent intracardiac electrophysiologic evaluation and, when possible, ambulatory monitoring before and after therapy with flecainide (mean dose 418 +/- 87 mg [mean +/- standard deviation]). An average of 4 antiarrhythmic agents were used and were unsuccessful before therapy with flecainide was begun. During 64 +/- 16 hours of control Holter monitoring in 16 patients, all had 1 or more salvos of VT, as well as ventricular premature complexes (VPCs). Programmed stimulation during the control period induced VT in 17 of 22 patients. After flecainide therapy, Holter monitoring showed elimination of all forms of VT in all but 1 patient, as well as significant reduction of paired VPCs by 95% (p less than 0.03) and single VPCs by 70% (p less than 0.005). Electrophysiologic study during flecainide therapy showed significant increases in AH, HV, PR, QRS and QTc intervals, and the ventricular effective refractory period. Programmed stimulation in 17 patients taking flecainide, with a mean plasma level of 1,075 +/- 521 ng/ml, showed ablation of inducible VT in only 2 patients, a worsening in 5 and continued VT inducibility in 10. Adverse effects that required drug withdrawal were infrequent and encountered in patients who received higher drug levels: 1 patient with congestive heart failure and 1 with severe sinus bradycardia. Thus, although flecainide suppresses complex ventricular arrhythmias on Holter recordings, it rarely alters the response to programmed stimulation. Caution is recommended in its use for recurrent sustained VT or VF and in the interpretation of electrophysiologic studies until the predictive value of programmed stimulation with flecainide therapy is established.     

Long-term outcome of patients with syncope associated with coronary artery disease and a nondiagnostic electrophysiologic evaluation

Syncope in the patient with structural heart disease and a nondiagnostic noninvasive workup is a generally accepted indication for an invasive electrophysiologic study. However, if the electrophysiologic evaluation is not highly sensitive, arrhythmic causes of syncope may not be discovered. In these patients, recurrent syncope and even sudden death may be observed at follow-up. Thus, we evaluated long-term follow-up in 68 consecutive patients who presented with syncope, coronary artery disease, and who had a negative invasive electrophysiologic evaluation. At a mean follow-up of 30 +/- 18 months (range 1 to 65), there have been 2 sudden deaths and 1 episode each of ventricular fibrillation and ventricular tachycardia in patients treated with an implantable cardioverter-defibrillator. All 4 arrhythmias occurred in patients with left ventricular fractions < or = 25%. Seventeen patients had recurrent presyncope or syncope. Bradycardia causing syncope was found in 8 of these patients. A bundle branch block at the initial evaluation predicted for the occurrence of bradycardia at follow-up. We conclude that in patients with coronary artery disease and syncope, noninducibility at electrophysiologic study predicts a lower risk of sudden death and ventricular arrhythmias. However, in patients with a reduced ejection fraction, the risk of sudden death and ventricular arrhythmias remains up to 10%/year and these patients may warrant treatment with implantable cardioverter-defibrillators. Recurrent syncope is common, and frequently a bradyarrhythmia is found to be the cause. Treatment of selected patients (especially those with bundle branch blocks) with permanent pacemakers may be justified.     

Diagnosis of norwalk virus infection by indirect enzyme immunoassay detection of salivary antibodies to recombinant norwalk virus antigen

Simple diagnostic tests are needed for the detection of norovirus (NoV) outbreaks. Salivary antibody assays provide an attractive alternative to collecting and testing serum or stool samples. Antibodies to Norwalk virus (NV) in oral fluid samples were compared with NV antibodies in serum collected from 38 volunteers challenged with NV inoculum. Pre- and postchallenge (day 4, 8, 14, and 21) saliva and serum samples were examined by enzyme immunoassay (EIA) using recombinant NV antigen. Of 18 infected subjects (those who shed NV in stool or who demonstrated immunoglobulin G [IgG] seroconversion), 15 (83%) had > or =4-fold increases in NV-specific salivary IgA and 15 (83%) had > or =4-fold increases in NV-specific salivary IgG when prechallenge and postchallenge saliva samples were compared. When the results of the IgA and IgG assays were combined, all 18 infected subjects showed > or =4-fold increases in NV-specific salivary IgG or IgA postchallenge titers compared to their prechallenge titers. One of 19 uninfected subjects had a > or =4-fold increase in NV-specific salivary IgG. The sensitivity of the combined assay results was 100%, and the specificity was 95%. NV-specific salivary IgA titers peaked around 14 days postchallenge. NV-specific salivary IgG and serum IgG titers continued to rise through 21 days postchallenge. The application of this EIA to an elementary school outbreak indicated that 67% of the subjects with confirmed infections had >4-fold rises in anti-NoV IgA when an antigen in the same genetic cluster as the outbreak virus was used. This is the first documented mucosal antibody response to NoV in children. This EIA provides a useful approach for diagnosing NoV outbreaks.     

Recommendations for the classification of group A rotaviruses using all 11 genomic RNA segments

Recently, a classification system was proposed for rotaviruses in which all the 11 genomic RNA segments are used (Matthijnssens et al. in J Virol 82:3204-3219, 2008). Based on nucleotide identity cut-off percentages, different genotypes were defined for each genome segment. A nomenclature for the comparison of complete rotavirus genomes was considered in which the notations Gx-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx are used for the VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5/6 encoding genes, respectively. This classification system is an extension of the previously applied genotype-based system which made use of the rotavirus gene segments encoding VP4, VP7, VP6, and NSP4. In order to assign rotavirus strains to one of the established genotypes or a new genotype, a standard procedure is proposed in this report. As more human and animal rotavirus genomes will be completely sequenced, new genotypes for each of the 11 gene segments may be identified. A Rotavirus Classification Working Group (RCWG) including specialists in molecular virology, infectious diseases, epidemiology, and public health was formed, which can assist in the appropriate delineation of new genotypes, thus avoiding duplications and helping minimize errors. Scientists discovering a potentially new rotavirus genotype for any of the 11 gene segments are invited to send the novel sequence to the RCWG, where the sequence will be analyzed, and a new nomenclature will be advised as appropriate. The RCWG will update the list of classified strains regularly and make this accessible on a website. Close collaboration with the Study Group Reoviridae of the International Committee on the Taxonomy of Viruses will be maintained.     

Head circumference and height in autism: a study by the Collaborative Program of Excellence in Autism

Data from 10 sites of the NICHD/NIDCD Collaborative Programs of Excellence in Autism were combined to study the distribution of head circumference and relationship to demographic and clinical variables. Three hundred thirty-eight probands with autism-spectrum disorder (ASD) including 208 probands with autism were studied along with 147 parents, 149 siblings, and typically developing controls. ASDs were diagnosed, and head circumference and clinical variables measured in a standardized manner across all sites. All subjects with autism met ADI-R, ADOS-G, DSM-IV, and ICD-10 criteria. The results show the distribution of standardized head circumference in autism is normal in shape, and the mean, variance, and rate of macrocephaly but not microcephaly are increased. Head circumference tends to be large relative to height in autism. No site, gender, age, SES, verbal, or non-verbal IQ effects were present in the autism sample. In addition to autism itself, standardized height and average parental head circumference were the most important factors predicting head circumference in individuals with autism. Mean standardized head circumference and rates of macrocephaly were similar in probands with autism and their parents. Increased head circumference was associated with a higher (more severe) ADI-R social algorithm score. Macrocephaly is associated with delayed onset of language. Although mean head circumference and rates of macrocephaly are increased in autism, a high degree of variability is present, underscoring the complex clinical heterogeneity of the disorder. The wide distribution of head circumference in autism has major implications for genetic, neuroimaging, and other neurobiological research.     

On chip cell separator using magnetic bead-based enrichment and depletion of various surface markers

This paper presents an on-chip magnetic cell sorting system for the sorting of cells based on a variety of surface markers. A polymer lab on a chip integrated with an electroplated array of Ni/Fe permalloy has been designed, fabricated, and characterized for the separation of cell substitutes at a variety of flow rates and incubation times. The system sequentially labels cell substitutes with magnetic beads and sorts them, repeating this process to sort for a variety of surface markers. Flow rates and incubation times were varied to characterize the system and produce the best combination of high specific capture and low nonspecific capture. The separation system developed on polymer is selective and efficient while being low cost, portable, and fabricated in a modular structure that can be integrated with other cell handling processes.