False positive of an immunochromatography kit for detection of norovirus in neonatal feces.

Norovirus was detected in the feces from five neonates in the growing care unit by a rapid immunochromatography (ICG) kit. However, confirmation using reverse transcription polymerase chain reaction (RT-PCR), RT-loop-mediated isothermal amplification (RT-LAMP), and nested RT-PCR methods showed negative results from all the feces. In addition, the ICG test for the detection of norovirus was positive for four cases out of the 16 feces from other asymptomatic neonates/infants. Only one feces out of the four samples was positive by RT-LAMP. In this study, among the factors related to false positives with the norovirus ICG kit, there were no differences regarding the commencement of feeding, nutrition, and sample collection methods. Since the false positive rate of ICG in the diagnosis of norovirus infection in neonates and early infancy is high, ICG is not an appropriate method, and it is necessary to confirm the results using reliable methods like RT-PCR.

The validity of the criteria for primary infection of Chlamydophila pneumoniae in children by measuring ELISA IgM antibodies.

As IgM antibody measurement by enzyme-linked immunosorbent assay (ELISA) has become possible for the serological diagnosis of Chlamydophila pneumoniae (C. pn) infection, the HITAZYME-ELISA method has become widely employed in Japan. However, in children, when the diagnostic criterion of primary infection is set at ID ≥1.1, the positive rate is higher than expected, and the potential for inaccurate reflection of the prevalence has been raised. In this study, we performed ROC analysis involving 136 pediatric patients with acute airway symptoms (0-14 years of age), considering a 32-fold or higher micro-immunofluorescence IgM antibody titer against C. pn as positive. Setting the cut-off value for ELISA C. pn IgM antibody ID at 2.0, the specificity was 100%, with no false positivity. The maximum (sensitivity + specificity)/2 was obtained when the cut-off value was set at 1.5. Therefore, IgM ID ≥2.0 was regarded as definitely positive and an IgM ID between 1.5 and 2.0 was regarded as indeterminate as diagnostic criteria for the primary infection. When the prevalence was investigated in 3,108 children (0-15 years of age) with airway symptoms based on these criteria, 542 cases (17.4%) were positive, and the median duration of IgM antibody positivity was five months. Long-term positivity (ten cases) for more than 12 months and recurrent positivity (eight cases) were also observed, but it may be appropriate to set a new criterion of IgM antibody ID ≥2.0 for the diagnosis of primary Chlamydophila pneumoniae infection in children.

Norovirus encephalopathy in a previously healthy child.

Norovirus causes acute gastroenteritis in all age groups. Afebrile convulsion is an occasional neurologic complication in norovirus infection, but encephalitis is rare. We report the case of a previously healthy 15-month-old girl with norovirus encephalopathy who had a poor neurologic outcome. Norovirus (genogroup II) was detected in plasma and stool by real-time reverse transcription polymerase chain reaction, but the cerebrospinal fluid showed negative result for genome. Elevated concentrations of cerebrospinal fluid interleukin-6, interleukin-10, interferon-γ, and tumor necrosis factor-α were observed on the third day of illness. The encephalopathy in our patient may be related to hypercytokinemia rather than to direct viral invasion.

[A clinical study of acute mastoiditis and mastoid lesions associated with pediatric otitis media].

Increased otitis media rendering acute mastoiditis and mastoid lesions severe or intractable appear to be related to dominant drug-resistant strains and the dissemination of nursery school attendance. Acute mastoiditis involves middle-ear inflammation spreading to the antrum mastoideum and accompanied by subcutaneous abscess. This emergency condition risks progression to subperiosteal abscess and meningitis. Mastoid cavity opacity in computed tomography (CT) scan often occurs with recurrent or intractable otitis media similar to that with mastoiditis. Four of the 8 cases of mastoiditis we treated were infant in whom upper respiratory tract pneumococcus and group A streptococcus were detected. Treatment involved antibiotics and myringotomy in all cases and surgery in two. Nine of the 10 cases of mastoid lesions with otitis media we saw were infant. All had pneumococcus detected, with accociated sinusitis.

Acute encephalopathy with biphasic seizures and late reduced diffusion associated with hemophagocytic syndrome.

We reported a girl with HHV-6 infection associated with both acute encephalopathy with biphasic seizures and late reduced diffusion, and hemophagocytic syndrome. She had a prolonged convulsion after a one-day history of febrile illness. Cerebrospinal fluid or brain CT showed no abnormalities on admission and her consciousness was recovered on the next day. However, a prolonged seizure and deterioration of consciousness appeared on the sixth day of illness. Diffusion-weighted images revealed marked reduction of water diffusion in the bilateral frontal areas. HHV-6 infection was virologically proven by polymerase chain reaction. She was treated with gamma-globulin, steroid pulse therapy, and brain hypothermia. In addition, decrease in white blood cells and platelet counts, and elevation of liver enzymes and ferritin were noted on the fourth day of illness. Hemophagocytic macrophages were revealed by bone marrow aspiration on the sixth day. Her hematological and blood chemistry abnormalities recovered gradually after steroid pulse therapy. An elevation of interleukin-6, -8, and -10, and tumor necrosis factor in the serum and that of interleukin-4, -6, and-8 in the cerebrospinal fluid were observed at the onset of a late seizure. These facts suggested that hypercytokinemia will be related to the pathogenesis of acute encephalopathy of our patient.

Prolonged Intracranial Hypertension after Recombinant Growth Hormone Therapy due to Impaired CSF Absorption.

We experienced a case of a Japanese boy who developed intractable idiopathic intracranial hypertension (IIH) during growth hormone (GH) treatment. At the age of 4 yr, the boy was diagnosed with idiopathic growth hormone deficiency, and recombinant human GH replacement was initiated. Nine months after initiation of the GH therapy, he began to complain of headache, but papilledema was not observed. His headache persisted thereafter, and right esotropia occurred 10 mo after the initiation of GH therapy, at which time papilledema was detected. No other neurological abnormalities were detected, and the findings of computed tomography and magnetic resonance imaging were normal. In a cerebrospinal fluid (CSF) examination, the pressure was markedly elevated to 450 mmH2O, but no other abnormality was recognized. Impaired CSF absorption was detected using the pressure-volume index technique. The CSF levels of GH and insulin-like growth factor I were not increased. GH therapy was withdrawn after it was suggested that the IIH was associated with the GH therapy, but the headache persisted. The intracranial hypertension did not respond to diuretics, and prednisolone was only transiently effective. Although the funduscopic findings were normalized, increased CSF pressure was still observed. For over 2 yr, repeated lumbar puncture was necessary to protect against visual defect. IIH is an uncommon adverse event during GH therapy, but it must be considered carefully.

[Discussion of positive initial infection criteria for Chlamydophila (Chlamydia) pneumoniae in children: IgM antibody titers by ELISA compared to micro-IF and Western blotting].

Microimmunofluorescence testing (micro-IF) is a standard serological test Chlamydia pneumoniae (C. pneumoniae) infection, but requires sophisticated techniques. ELISA, a simple, easy diagnostic method, has thus come into wide use and is included by the National Insurance System of Japan as an approved and reimbursable procedure. Based on Kishimoto's criteria, initial C. pneumoniae infection is diagnosed by ELISA at single serum IgM antibody titer in children ID > or = 1.10, adult ID > or = 1.60. The positive percentage of C. pneumoniae IgM antibody in children, however, is unexpectedly high, and the antibody level has been found to remain positive for several months or more. In an effort to revise positive criteria for initial infection with C. pneumoniae, we compared IgM antibody titer of ELISA to that of micro-IF and Western blotting (WB) in acute pediatric respiratory tract infection. Specimens were collected from 128 children with acute respiratory tract infection; 106 (11-months to 14-year-olds) with an IgM antibody ID > or = 1.10 and 22 (8-months to 12-year-olds) with an ID < 1.10. The 77 samples with an ID > or = 1.40 of IgM antibody by ELISA included 4 negatives in micro-IF and 8 negatives in WB. Regarding definitive cases as C. pneumoniae-IgM positive by both micro-IF and WB, the receiver operating characteristic curve showed that the optimal value was ELISA ID 1.40; sensitivity 86.8%, specificity 96.3%, the positive predictive value 98.5%, and the negative predictive value 72.2%. Diagnostic precision in initial C. pneumoniae infection in children may therefore be improved by revising positive criteria of ELISA IgM titer.

ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe.

Cloning of the entire set of an organism's protein-coding open reading frames (ORFs), or 'ORFeome', is a means of connecting the genome to downstream 'omics' applications. Here we report a proteome-scale study of the fission yeast Schizosaccharomyces pombe based on cloning of the ORFeome. Taking advantage of a recombination-based cloning system, we obtained 4,910 ORFs in a form that is readily usable in various analyses. First, we evaluated ORF prediction in the fission yeast genome project by expressing each ORF tagged at the 3' terminus. Next, we determined the localization of 4,431 proteins, corresponding to approximately 90% of the fission yeast proteome, by tagging each ORF with the yellow fluorescent protein. Furthermore, using leptomycin B, an inhibitor of the nuclear export protein Crm1, we identified 285 proteins whose localization is regulated by Crm1.

pDUAL, a multipurpose, multicopy vector capable of chromosomal integration in fission yeast.

A novel series of plasmid vectors named pDUAL have been developed. These vectors enable one to introduce not only multicopies of genes with episomal maintenance but also a single copy with chromosomal integration into the fission yeast, Schizosaccharomyces pombe. The multicopy plasmids can be easily converted to fragments for chromosomal integration by digestion of the plasmids with a certain restriction endonuclease before transformation of the yeast cells. The resultant fragments, lacking the autonomously replicating sequence, are designed for targeting into the chromosomal leu1 locus by homologous recombination. Whether the transformants are the results of episomal maintenance of the plasmid or homologous gene targeting can be readily checked by their requirement for uracil or leucine, or by the PCR diagnostic analysis. Furthermore, we propose the use of pDUAL derivatives for PCR-based chromosomal tagging of a gene to introduce several tags into 5'-terminus of a gene, employing a set of primers. Using these all-in-one vectors, a suitable mode of expression of a cloned gene can be selected for individual analysis without any complicated subcloning processes.