Noriko Kubota

First isolation of oleate-dependent Enterococcus faecalis small-colony variants from the umbilical exudate of a paediatric patient with omphalitis

An oleate-dependent Enterococcus faecalis isolate representing small-colony variants (SCVs) was isolated from the umbilical exudate of a 31-month-old Japanese male patient in Nagano Childrens Hospital, Azumino, Japan. The patient had been suffering from recurrent omphalitis since early infancy. The initial E. faecalis SCV isolate formed small colonies on sheep blood agar plates and tiny colonies on chocolate and modified Drigalski agar, although no visible growth was observed in HK-semi solid medium after 48 h incubation in ambient air. Moreover, the SCV isolate, the colonial morphology of which was reminiscent of Streptococcus species, could not be identified using the MicroScan WalkAway-40 and API 20 Strep systems, both of which yielded profile numbers that did not correspond to any bacterial species, probably as a result of insufficient growth of the isolate. The SCV isolate was subsequently identified as E. faecalis based on its morphological, cultural and biochemical properties, and this was confirmed by sequencing the 16S rRNA gene of the organism. Investigations revealed that the addition of oleate, an unsaturated fatty acid, enabled the isolate to grow on every medium with normal-sized colony morphology. Although it has long been known that long-chain fatty acids, especially unsaturated oleic acid, have a major inhibitory effect on the growth of a variety of microorganisms, including not only mycobacteria but also streptococci, this is, to the best of our knowledge, the first clinical isolation of an oleate-dependent E. faecalis SCV isolate. In addition, oleic acid might be considered to affect the cell membrane permeability of carbohydrates or antimicrobial agents such as β-lactams.

Complete hydatidiform mole in a triplet pregnancy coexisting two viable fetuses: case report and review of the literature.

We report a rare case of a complete hydatidiform mole with two or more coexisting fetuses where both infants survived without complications. A male infant weighing 1258 g and a female infant weighing 880 g were delivered without complications and discharged 95 days after the birth. The analysis of DNA microsatellite polymorphisms indicated that the mole was of paternal origin and probably homozygous. The mother suffered from multiple pulmonary metastasis of the hydatidiform mole which was detected 3 days after the surgery and was successfully treated with methotrexate. A complete hydatidiform mole with two or more coexisting fetuses produces a dilemma between immediate termination and pregnancy continuation. Although the present case resulted in a favorable outcome, a review of the 14 reported cases suggests that the high fetal loss rate (90%) must be a consideration in the decision regarding management of such a pregnancy.

Exome sequencing-based identification of mutations in non-syndromic genes among individuals with apparently syndromic features

In a clinical setting, the number of organ systems involved is crucial for the differential diagnosis of congenital genetic disorders. When more than one organ system is involved, a syndromic diagnosis is suspected. In this report, we describe three patients with apparently syndromic features. Exome sequencing identified non‐syndromic gene mutations as a potential cause of part of their phenotype. The first patient (Patient 1) is a girl with cleft lip/palate, meningoencephalocele, tetralogy of Fallot, and developmental delay. The second and third patients (Patients 2 and 3) are brothers with developmental delay, deafness, and low bone mineral density. Exome sequencing revealed the presence of a CDH1 mutation in Patient 1 and a PLS3 mutation in Patients 2 and 3. CDH1 mutations are known to be associated with non‐syndromic cleft lip/palate, while PLS3 mutations are associated with osteoporosis. Thus, these variants may explain a part of the complex phenotype of the patients, although the effects of these missense variants need to be evaluated by functional assays in order to prove pathogenicity. On the basis of these findings, we emphasize the importance of scrutinizing non‐syndromic gene mutations even in individuals with apparently syndromic features.

Perfluorocarbon Suppresses Lipopolysaccharide- and α-Toxin-Induced Interleukin-8 Release from Alveolar Epithelial Cells

Background/Aims: Human pulmonary alveolar epithelial (A549) cells release interleukin-8 (IL-8) on stimulation by lipopolysaccharide (LPS) and α-toxin. We hypothesised that the perfluorocarbons (PFCs), perflubron and FC-84, would block stimulation of A549 cells by these toxins. Methods: The levels of IL-8 production in A549 cells were measured following exposure to toxins for 24 h with or without PFC. The amount of IL-8 released from A549 cells was measured by enzyme-linked immunosorbent assay, and the level of IL-8 mRNA was measured by real-time RT-PCR. Results: When stimulated with LPS or α-toxin, IL-8 release from A549 cells increased. There were no significant differences in level of IL-8 release between cells pre-incubated for 24 h with or without PFC after toxin stimulation for 24 h. When PFC was administered along with LPS stimulation, the level of IL-8 release was decreased (LPS control, 1,398 ± 110 pg/well; FC-84, 686 ± 50 pg/well; perflubron, 749 ± 137 pg/well; p < 0.05). Levels of IL-8 mRNA expression were significantly higher with than without LPS, and those with LPS and perflubron were significantly lower than those with LPS alone. Conclusions: The results show that PFCs block stimulation of A549 cells by LPS or α-toxin. PFC may be useful clinically in treatment of pulmonary inflammation in the alveolar space.

Notable alkaline tolerance of Kocuria marina isolate from blood of a pediatric patient with continuous intravenous epoprostenol therapy

This study was the first to describe the hitherto deficiently evaluated alkaline tolerance of Kocuria marina isolate from a pediatric patient with continuous intravenous epoprostenol dosing therapy. Our isolate from blood of a 7-year-old Japanese boy was finally identified as K. marina by the morphological, cultural, and biochemical properties together with the comparative sequence analyses of the 16S rRNA genes. The K. marina isolate, the causative agent of catheter-related blood-stream infection, was not only revealed to be salt tolerant (NaCl 15%), but also demonstrated to be stably survived with no apparent decrease of cell counts for long periods (120 h) in an alkaline environment (pH 8, 9, 10, and 11) at 35 °C. Its remarkable tolerance to the stresses of high alkalinity compared with a clinical Staphylococcus aureus strain should provide consistent interpretation that the environment of high alkalinity (pH 10.2-10.8) measures should be insufficient to inactivate almost all the causative agents including K. marina strains in the solution of epoprostenol (pH 10.4) (Flolan(®), GlaxoSmithKline, Ltd., Tokyo, Japan.). To the best of our knowledge, the first description of the property of being tolerant to high alkalinity that the K. marina isolate exhibited was noteworthy and a useful piece of information. In conclusion, we believe that the present study should be a notification regarding the potential risk of catheter-related blood-stream infections due to K. marina, suggestive of an alkalophile, especially in patients receiving continuous intravenous epoprostenol dosing therapy.

Successful cessation of transmitting healthcare -- associated infections due to Burkholderia cepacia complex in a neonatal intensive care unit in a Japanese children's hospital.

BackgroundBurkholderia cepacia strains have been known to possess the capability to cause serious infections especially in neonatal intensive care units (NICUs), and their multi-drug resistances become a severe threat in hospital settings. The aim of this investigation was to evaluate the B. cepacia complex infections in the NICU in Nagano Childrens Hospital, Azumino 399-8288, Japan, and to report the intervention leading to the successful cessation of the outbreak.MethodologyThe incidence of isolation and antimicrobial susceptibilities of nosocomial Burkholderia cepacia complex strains during a four-year period were retrospectively examined by clinical microbiological records, and by pulsed-field gel electrophoresis analyses along with the bacteriological verification of disinfectant device itself and procedures for its maintenance routinely used in the NICU.ResultsDuring the period surveyed between 2007 and 2009, only an isolate per respective year of B. cepacia complex was recovered from each neonate in the NICU. However, in 2010, the successive 6 B. cepacia complex isolates were recovered from different hospitalized neonates. Among them, an isolate was originated from peripheral blood of a neonate, apparently giving rise to systemic infection. In addition, the hospitalized neonate with bacteremia due to B. cepacia complex also exhibited positive cultures from repeated catheterized urine samples together with tracheal aspirate secretions. However other 5 isolates were considered as the transients or contaminants having little to do with infections. Moreover, the 5 isolates between July and October in 2010 revealed completely the same electrophoresis patterns by means of pulsed-field gel electrophoresis analyses, strongly indicating that they were infected through the same medical practices, or by transmission of the same contaminant.ConclusionsA small outbreak due to B. cepacia complex was brought about in the NICU in 2010, which appeared to be associated with the same genomovar of B. cepacia complex. The source or the rout of infection was unknown in spite of the repeated epidemiological investigation. It is noteworthy that no outbreak due to B. cepacia complex was noted in the NICU after extensive surveillance intervention.

Discordant clinical phenotype in monozygotic twins with Alagille syndrome: Possible influence of non-genetic factors.

Alagille syndrome is a multisystem developmental disorder characterized by bile duct paucity, congenital heart disease, vertebral anomalies, posterior embryotoxon, and characteristic facial features. Alagille syndrome is typically the result of germline mutations in JAG1 or NOTCH2 and is one of several human diseases caused by Notch signaling abnormalities. A wide phenotypic spectrum has been well documented in Alagille syndrome. Therefore, monozygotic twins with Alagille syndrome provide a unique opportunity to evaluate potential phenotypic modifiers such as environmental factors or stochastic effects of gene expression. In this report, we describe an Alagille syndrome monozygotic twin pair with discordant placental and clinical findings. We propose that environmental factors such as prenatal hypoxia may have played a role in determining the phenotypic severity.

Pretransplant-corrected QT dispersion as a predictor of pericardial effusion after pediatric hematopoietic stem cell transplantation

Pericardial effusion is a potentially fatal complication following hematopoietic stem cell transplantation (HSCT). Therefore, the identification of risk factors could improve the outcome. Prolonged QT dispersion (QTD) and corrected QTD (QTcD) are associated with serious arrhythmias and sudden death in many forms of heart disease. However, no study has evaluated the efficacy of QTD and QTcD to predict pericardial effusion post‐HSCT. We studied 89 pediatric HSCT patients to identify preoperative risk factors for pericardial effusion with particular focus on QTD and QTcD. Pericardial effusion occurred in 15 patients (cumulative onset rate: 17.4%) within one year post‐HSCT, of which 8 (9.2%) were symptomatic. Patients with pericardial effusion following allogeneic HSCT showed significantly lower overall survival; however, pericardial effusion was not the direct cause of death in any patient. Univariate and multivariate analyses revealed that transplantation‐associated thrombotic microangiopathy (TA‐TMA) was an independent risk factor for post‐HSCT pericardial effusion. In addition, pretransplant QTcD was significantly prolonged in the pericardial effusion group. These results suggest that pediatric patients with abnormally prolonged QTcD before the preparative regimen for HSCT should be regularly followed‐up by echocardiography to detect pericardial effusion, particularly when accompanied by complications including TA‐TMA.

Dissecting the phenotype of supernumerary marker chromosome 20 in a patient with syndromic pierre robin sequence: Combinatorial effect of gene dosage and uniparental disomy

Clinical phenotypes in individuals with a supernumerary marker chromosome (SMC) are mainly caused by gene dosage effects due to the genes located on the SMC. An additional effect may result from uniparental disomy (UPD). Consequently, the occurrence of UPD may be a confounding factor in identifying genotype‐phenotype correlations in SMC syndromes. Here, we report on a patient that illustrates this problem; the phenotype of this patient was a consequence of a combined effect of gene dosage and UPD. The proband showed facial dysmorphisms, growth retardation and developmental delay. G‐band karyotype of the probands peripheral blood showed the presence of mosaic SMC. A SNP array analysis documented maternal UPD20 and 20p duplication. It is known that maternal UPD20 causes prenatal onset growth retardation and feeding difficulties. By contrast, duplication of 20p causes facial dysmorphisms, micrognathia, cleft palate, developmental delay and vertebral anomalies. Our classification of the probands phenotype showed a mixture of these two effects. Therefore, we suggest the routine use of genome‐wide SNP array towards the detailed genotype‐phenotype correlations for SMC syndromes.

Case report of a 6-year-old girl with Mycoplasma hominis ventriculoperitoneal shunt infection

Mycoplasma hominis is a rare causative pathogen for surgical site infections after neurosurgical procedures. This organism lacks a cell wall, rendering it undetectable by Gram staining and making it resistant to beta-lactam antibiotics. In addition, some special techniques are required to identify this organism. Thus, it is very difficult to diagnose infections caused by this pathogen. Here, the authors report a pediatric case of M. hominis ventriculoperitoneal shunt (VPS) infection with central nervous system involvement for which beta-lactam antibiotics were not effective and Gram staining revealed no pathogens. Because few cases have been described that involve the treatment of M. hominis infection after neurosurgery, in this case the patients serum and CSF were monitored for antibiotic drug concentrations. Successful treatment of the infection was achieved after approximately 6 weeks of administration of clindamycin and ciprofloxacin antibiotics in addition to external ventricular drain revision and subsequent VPS replacement. When beta-lactam antibiotics are ineffective and when Gram staining cannot detect the responsible pathogens, it is important to consider M. hominis as the atypical pathogen.