Automated detection of outbreaks of antimicrobial-resistant bacteria in Japan.

BACKGROUND: Hospital outbreaks of antimicrobial-resistant (AMR) bacteria should be detected and controlled as early as possible. AIM: To develop a framework for automatic detection of AMR outbreaks in hospitals. METHODS: Japan Nosocomial Infections Surveillance (JANIS) is one of the largest national AMR surveillance systems in the world. For this study, all bacterial data in the JANIS database were extracted between 2011 and 2016. WHONET, a free software for the management of microbiology data, and SaTScan, a free cluster detection tool embedded in WHONET, were used to analyse 2015-2016 data of eligible hospitals. Manual evaluation and validation of 10 representative hospitals around Japan were then performed using 2011-2016 data. FINDINGS: Data from 1031 hospitals were studied; mid-sized (200-499 beds) hospitals accounted for 60%, followed by large hospitals (≥500 beds; 24%) and small hospitals (<200 beds; 16%). More clusters were detected in large hospitals. Most of the clusters included five or fewer patients. From the in-depth analysis of 10 hospitals, ∼80% of the detected clusters were unrecognized by infection control staff because the bacterial species involved were not included in the priority pathogen list for routine surveillance. In two hospitals, clusters of more susceptible isolates were detected before outbreaks of more resistant pathogens. CONCLUSION: WHONET-SaTScan can automatically detect clusters of epidemiologically related patients based on isolate resistance profiles beyond lists of high-priority AMR pathogens. If clusters of more susceptible isolates can be detected, it may allow early intervention in infection control practices before outbreaks of more resistant pathogens occur.

Clock gene dysfunction in patients with obstructive sleep apnoea syndrome.

Clock genes regulate mammalian circadian rhythms, and dysfunction of clock genes can contribute to various disorders. To investigate whether obstructive sleep apnoea syndrome (OSAS) influences clock gene function, the present authors examined Period1 (Per1) mRNA expression in vitro and in vivo. In eight healthy subjects and eight OSAS patients, plasma noradrenaline, serum interleukin (IL)-6, high-sensitivity C-reactive protein (hsCRP) and Per1 mRNA expression in peripheral whole blood were measured. Expression of Per1 mRNA in cultured cells was examined under IL-6 or noradrenaline stimulation in vitro. After noradrenaline was administered to mice in vivo, Per1 mRNA expression in the brain was examined. The concentrations of serum IL-6, hsCRP and plasma noradrenaline were elevated in OSAS patients, but improved by continuous positive airway pressure (CPAP) therapy. Per1 mRNA expression in the peripheral blood significantly decreased at 02:00 h by CPAP in OSAS patients. Stimulation with IL-6 did not directly induce Per1 mRNA in vitro. Administration of noradrenaline induced Per1 mRNA in the cerebral cortex of mice in vivo. The current study revealed that obstructive sleep apnoea syndrome caused clock gene dysfunction, and continuous positive airway pressure helped to improve it. Sympathetic activation and elevation of the plasma noradrenaline concentration in obstructive sleep apnoea syndrome may be one of the factors involved in disorders of Period1 mRNA expression.

Structure of the RGS-like domain from PDZ-RhoGEF: linking heterotrimeric g protein-coupled signaling to Rho GTPases.

BACKGROUND: The multidomain PDZ-RhoGEF is one of many known guanine nucleotide exchange factors that upregulate Rho GTPases. PDZ-RhoGEF and related family members play a critical role in a molecular signaling pathway from heterotrimeric G protein-coupled receptors to Rho proteins. A approximately 200 residue RGS-like (RGSL) domain in PDZ-RhoGEF and its homologs is responsible for the direct association with Galpha12/13 proteins. To better understand structure-function relationships, we initiated crystallographic studies of the RGSL domain from human PDZ-RhoGEF. RESULTS: A recombinant construct of the RGSL domain was expressed in Escherichia coli and purified, but it did not crystallize. Alternative constructs were designed based on a novel strategy of targeting lysine and glutamic acid residues for mutagenesis to alanine. A triple-point mutant functionally identical to the wild-type protein was crystallized, and its structure was determined by the MAD method using Se-methionine (Se-Met) incorporation. A molecular model of the RGSL domain was refined at 2.2 A resolution, revealing an all-helical tertiary fold with the mutations located at intermolecular lattice contacts. CONCLUSIONS: The first nine helices adopt a fold similar to that observed for RGS proteins, although the sequence identity with other such known structures is below 20%. The last three helices are an integral extension of the RGS fold, packing tightly against helices 3 and 4 with multiple hydrophobic interactions. Comparison with RGS proteins suggests features that are likely relevant for interaction with G proteins. Finally, we conclude that the strategy used to produce crystals was beneficial and might be applicable to other proteins resistant to crystallization.

Leukemia-associated Rho guanine nucleotide exchange factor (LARG) links heterotrimeric G proteins of the G(12) family to Rho.

A putative guanine nucleotide exchange factor (GEF), termed leukemia-associated RhoGEF (LARG), was recently identified upon fusion to the coding sequence of the MLL gene in acute myeloid leukemia. Although the function of LARG is still unknown, it exhibits a number of structural domains suggestive of a role in signal transduction, including a PDZ domain, a LH/RGS domain, and a Dbl homology/pleckstrin homology domain. Here, we show that LARG can activate Rho in vivo. Furthermore, we present evidence that LARG is an integral component of a novel biochemical route whereby G protein-coupled receptors (GPCRs) and heterotrimeric G proteins of the G alpha(12) family stimulate Rho-dependent signaling pathways.

A novel interstitial deletion of KAL1 in a Japanese family with Kallmann syndrome.

We identified a novel interstitial deletion that spanned from exons 5 to 10 of KAL1 in two Japanese brothers with X-linked Kallmann syndrome (KS; MIM no. 308700). Both brothers had hypogonadism, unilateral renal agenesis, and disturbance of the sense of smell, but they had no other neurological manifestations, including mental disturbance. Their mother was confirmed to be an asymptomatic carrier, by use of a comparative multiplex polymerase chain reaction (PCR) analysis. The present patients are further examples of patients with KS without mental disturbance caused by a mutation confined to KAL1.

Clinical and in vitro evaluation of membrane humidifier that does not require addition of water.

It is well known that conventional bubbling humidifiers are capable of producing micro-aerosols contaminated with bacteria. We developed a unique humidifier, named a membrane humidifier, that does not require an external water supply. This new system obtains moisture from room air. We investigated the clinical and in vitro evaluation of the membrane humidifier. Ten patients with chronic pulmonary disease participated in the study. We evaluated the partial pressure of oxygen in arterial blood (PaO2) of 10 patients who used the new device. We conducted an in vitro study to determine whether the device could prevent the bacterial contamination of humidified-oxygen. We passed compressed air contaminated with Pseudomonas aeruginosa outside the hollow fibres of the membrane humidifier, and the humidified-oxygen passed inside the hollow fibres was sampled into nutrient broth periodically for 10 days. We also compared the relative humidity of oxygen humidified by a membrane humidifier with that of oxygen humidified by a bubbling humidifier. There was no significant difference between measured PaO2 while breathing oxygen humidified using a membrane humidifier and that while breathing oxygen humidified using a bubbling humidifier. Cultures of the humidified-oxygen passed through the hollow fibres were negative for bacteria. The membrane humidifier could produce good humidification. The new device appeared to prevent bacterial contamination, and may help to reduce the risk of infection in patients at hospital and home.

Fibrillin gene (FBN1) mutations in Japanese patients with Marfan syndrome.

Marfan syndrome (MFS; MIM #154700) is a connective tissue disorder characterized by cardiovascular, skeletal, and ocular abnormalities. The fibrillin-1 gene (FBN1; MIM no. 134797) on chromosome 15 was revealed to be the cause of Marfan syndrome. To date over 137 types of FBN1 mutations have been reported. In this study, two novel mutations and a recurrent de-novo mutation were identified in patients with MFS by means of single-strand conformational polymorphism (SSCP) analysis. The two novel mutations are a 4-bp deletion at nucleotide 2820-2823 and a G-to-T transversion at nucleotide 1421 (C474F), located on exon 23 and exon 11, respectively. A previously reported mutation at the splicing donor site of intron 2 (IVS2 G + 1A), which is predicted to cause exon skipping, was identified in a sporadic patient with classical MFS.

Functional assay of NF-kappaB translocation into nuclei by laser scanning cytometry: inhibitory effect by dexamethasone or theophylline.

Nuclear factor-kappa B (NF-kappaB)/Rel transcription factors play an important role in the inducible regulation of a variety of cytokine genes in epithelial cells. We assessed accumulation of fluorescence-stained NF-kappaB into propidium iodide-stained nuclei using laser scanning cytometry. "Activity-specific" antibodies to the Rel A (p65) and NF-kappaB1 (p50) subunits of NF-kappaB were detected in the nuclei of A549 cells (an immortalized human type II alveolar epithelial cell line). Exposure to TNF-alpha caused p65 and p50 to translocate into nuclei in a dose- and time-dependent manner. Preincubation with dexamethasone for 24 h or 30 min, or with theophylline for 30 min before TNF-alpha stimulation attenuated the nuclear translocation of Rel A and p50. These findings suggest that theophylline, as well as dexamethasone, may inhibit translocation of NF-kappaB.

[Relationship between cancer chemotherapeutic drug-induced delayed emesis and plasma levels of substance P in two patients with small cell lung cancer].

We investigated the relationship between delayed emesis caused by cisplatin (CDDP) based chemotherapy and plasma levels of serotonin, 5-hydroxy-indoleacetic acid (5-HIAA), and substance P in two patients with small cell lung cancer. In each of the cases, we used the 5-HT3 receptor antagonist ramosetron every morning on day 1-4 of the chemotherapy. In case 1, plasma levels of serotonin were low, whereas substance P levels increased since 24 hours after the injection of CDDP. The increase in substance P levels paralleled the onset of vomiting. In this case, however, substance P levels decreased and yet vomiting occurred. Similarly, in case 2, plasma levels of serotonin were low, whereas substance P levels increased since 24 hours. The increase in plasma levels of substance P and the onset of vomiting were observed at the same time 2-3 days after cisplatin administration. In this case, however, vomiting was not observed during the 5 days when the substance P was highest. Therefore, we suggested that substance P was closely associated with CDDP-induced delayed emesis, though some chemical mediators other than substance P might also be related to the emesis.

Clinical efficacy of the FLUTTER device for airway mucus clearance in patients with diffuse panbronchiolitis.

Expectoration of mucus is important in preventing the development of airway inflammation in patients with diffuse panbronchiolitis (DPB). To evaluate the clinical efficacy of the FLUTTER device in clearing mucus from the airways of patients with DPB who have difficulty expectorating, we assessed pulmonary function and symptoms in patients treated with FLUTTER. Eight patients in a stable clinical condition with DPB were included in the study. The study was divided into two consecutive, 1-week periods. The initial week was an observation week. During the following week, patients used FLUTTER four times daily. Expectorated sputum was collected in a container and weighed every day during 2 weeks. Pulmonary function, partial oxygen pressure and partial carbon dioxide pressure in arterial blood were measured in all patients on the last day of the observation week and the FLUTTER treatment week. A symptom score for difficulty of expectoration was determined by questionnaire. A pneumothorax developed in one patient during using FLUTTER. The mean daily sputum weight and peak expiratory flow rate increased significantly after treatment with FLUTTER ( P< 0.04 and P< 0.02, respectively). Symptom score improved significantly after using FLUTTER ( P< 0.02). We conclude that the use of FLUTTER is effective in clearing mucus from the airways. However, the development of a pneumothorax may complicate use of the procedure in some cases.

Efficacy of newly developed pressure swing adsorption type oxygen concentrator with membrane humidifier: comparison with conventional oxygen concentrator with bubble water humidifier.

To examine the clinical efficacy of a newly developed pressure swing adsorption (PSA) type oxygen concentrator with a membrane humidifier without added water for humidification, the new machine was compared with the conventional PSA type oxygen concentrator with bubble water humidifier in 10 patients with chronic pulmonary disease. Relative humidity, partial pressure of oxygen (PaO2) and partial pressure of carbon dioxide (PaCO2) in arterial blood were measured when the patient breathed air and oxygen from the oxygen concentrators. No significant difference between PaO2 while breathing oxygen flow from the new machine and that while breathing oxygen from the conventional oxygen concentrator was observed. All patients answered that there was no difference on subjective impression between breathing oxygen from the new machine and from the conventional oxygen concentrator. Sufficient relative humidity (above 50%) of oxygen flow was obtained by using membrane humidifier. Since this machine saves the troublesome procedures of cleaning the container and changing the water, it will be beneficial to the patients who use a PSA-type oxygen concentrator in their home.

[Assay of specific anti-Chlamydia pneumoniae antibodies by ELISA method. 2. studies on clinical usefulness and serological diagnostic standards].

We measured anti-Chlamydia pneumoniae (C. pneumoniae) specific antibody titers by means of a newly-developed enzyme-linked immunosorbent assay (ELISA) method using an anti-C. pneumoniae specific antibody detection reagent. The clinical usefulness of this method was hereby evaluated. The IgG, IgA and IgM titers in 418 serum specimens obtained from patients with respiratory tract infections were measured by this new ELISA method, and the results were compared with the titers determined for the same specimens with the micro immunofluorescence (Micro-IF) method. The results showed good correlation coefficients for IgG, IgA and IgM. The two assay methods showed high agreement rates for positivity and for negativity. Specimens which did not yield the same results with the ELISA method and the Micro-IF method were subjected to analysis by the Western blot method, and the rates of agreement with the ELISA results were high. In addition, the child (0 approximately 15 yrs old; n = 122) and adult (16 approximately 90 yrs old; n = 133) cases were classified on the basis of being antigen-positive or antigen-negative at the initial examination, and their antibody-positive rates were determined. The adults showed no statistically significant differences in the antibody-positive rates for either IgG or IgA antibodies as a function of the pretreatment antigen status. However, the children showed statistically significant (p < 0.001) differences in the antibody-positive rates for both IgG and IgA antibodies as a function of the antigen status in the antigen-positive group compared with the rates in the antigen-negative group. Furthermore, the IgM-positive rates for the children were high in the antigen-positive group compared with the rates in the antigen-negative group, and the difference was statistically significant (p < 0.001). The IgM-positive rates in the adults were also significantly (p < 0.05) different between the antigen-positive group and the antigen-negative group. The Micro-IF method was applied to 34 specimens from antigen-positive patients, and 22 specimens were found to show an IgG titer of > or = 512 or an IgM titer of > or = 16. The diagnoses of these patients were acute respiratory disease in sixteen, pneumonia in four. Application of the ELISA-method to those 22 specimens showed all of them to exhibit IgG absorbance of > or = 0.6 and IgA absorbance of 0.2. The results described above indicate the clinical usefulness of our new ELISA method for the detection of antibodies specific for C. pneumoniae. The significance of this ELISA method for serological diagnosis of C. pneumoniae infections and the criteria for diagnosis of acute infections were also discussed.

[A case of adult Chlamydia pneumoniae pneumonia diagnosed from a culture method].

We diagnosed a 41-year-old female patient to be suffering from Chlamydia pneumoniae (C. pneumoniae) by using PCR and culture methods. She had a prolonged dry cough and slight fever. Her chest roentgenogram showed a segmental infiltration in the middle of the right lung field. We treated her with 400 mg of cefpodoxime proxetil (CPDX-PR) per day. On the 4th day after beginning the treatment with CPDX-PR, she still complained of a productive cough. We changed the treatment by using 300 mg of roxithromycin per day and these symptoms disappeared. To diagnose C. pneumoniae early, PCR, MIF and culture methods are very useful diagnostic tools.