Legionella prevalence and risk of legionellosis in Japanese households.

This study determined the occurrence of legionellae in private houses for which there were no available data on aquatic environments other than the water supply system. From June 2013 to November 2014, we collected 138 water and 90 swab samples from aquatic environments in 19 houses. Legionella DNA was detected via a loop-mediated isothermal amplification assay in 66 (47·8%) water and 17 (18·9%) swab samples. High Legionella DNA detection rates were observed in water samples from washing machines and aquariums. Legionella spp. was isolated from 9 (6·5%) water and 3 (3·3%) swab samples. Legionella pneumophila SG 1 was detected from the outlet water of a bathtub spout and a bath sponge. Use of amoebic co-culture effectively increased legionellae and Legionella DNA detection rates from all sample types. A logistic regression analysis revealed that the heterotrophic plate count was significantly related to Legionella contamination. Our findings indicate that there is a risk of legionellosis from exposure to Legionella spp. in a variety of aquatic environments in residential houses. Control measures for legionellae in houses should include frequent cleaning and disinfecting to reduce heterotrophic bacteria in water and, where possible, preventing aerosolization from aquatic environments.

Outbreak of Legionnaires' disease on a cruise ship linked to spa-bath filter stones contaminated with Legionella pneumophila serogroup 5.

In January 2003, two cases of Legionnaires' disease associated with a ship's cruise were registered in the database of National Epidemiological Surveillance of Infectious Diseases. A 70-year-old male heavy smoker with mild emphysema contracted the disease during a cruise. Legionella pneumophila serogroup (sg) 5 was isolated from the patient's sputum and the ship's indoor spa. The isolate from the spa matched the patient's isolate by genotyping performed by pulsed-field gel electrophoresis (PFGE). The second case was in a 73-year-old female. During epidemiological investigation, a third case of Legionnaire's disease in a 71-year-old male was subsequently diagnosed among passengers on the same ship on the following cruise. Environmental investigation revealed that porous natural stones (Maifanshi) in the filters of the spas had harboured L. pneumophila, a phenomenon which has not been reported except in Japan. This is the first documented evidence of L. pneumophila sg 5 infection on a ship and of porous stones as a source of Legionella infection.

Development status of Terumo implantable left ventricular assist system.

We have been developing an implantable left ventricular assist system (T-ILVAS) featuring a magnetically suspended centrifugal pump (MSCP) since 1995. In vitro and in vivo studies using a prototype MSCP composed of a polycarbonate housing and impeller (196 ml) have demonstrated long-term durability and excellent blood compatibility for up to 864 days, and excellent stability of the magnetic bearing of the MSCP. These preliminary results strongly suggested that the magnetic bearing of the MSCP is reliable and is a most feasible mechanism for a long-term circulatory assist device. We have recently devised a clinical version pump made of titanium (180 ml) with a new position sensor mechanism and a wearable controller with batteries. Cadaver fit study confirmed that the Type IV pump could be implanted in a small patient with a body surface area as small as 1.3. The in vitro performance tests of the Type IV pump demonstrated excellent hydrodynamic performances with an acceptable hemolysis rate. New position sensors for the titanium housing showed more uniform sensor outputs of a magnetic bearing than in the prototype polycarbonate pump. The Type IV pump then was evaluated in vivo in 6 sheep at the Oxford Heart Centre. Four sheep were electively sacrificed at 3 months and were allowed to survive for more than 6 months for long-term evaluation. In this particular series of experiments, no anticoagulant/antiplatelet regimen was utilized except for a bolus dose of heparin during surgery. There was a left ventricular mural thrombi around the inflow cannula in 1 sheep. Otherwise, there was no mechanical failure nor sign of thromboembolism throughout the study.

Terumo implantable left ventricular assist system: results of long-term animal study.

The research group of Terumo Corporation, NTN Corporation, and the Setsunan University have been developing an implantable left ventricular assist system (T-ILVAS) featuring a centrifugal blood pump with a magnetically suspended impeller (MSCP). The present study describes results of chronic animal experiments using the MSCP. The MSCP has been tested ex vivo and in vivo in 6 sheep as a left heart bypass between the left ventricular apex and descending aorta. Ex vivo chronic sheep experiments using Model I demonstrated long-term durability, nonthrombogenicity, low hemolysis (<6 mg/dl), and excellent stability of the magnetic bearing with long-term survival for up to 864 days. Average pump flow rate was 4 L/min at a fixed rotational speed of 2000 rpm. Power spectral analyses of heart rate, aortic pressure, and blood temperature maintained normal 1/f fluctuation during the study. The retrieved pump was completely free from thrombus formation and there was no evidence of infarct in major organs. The implantable Model II was evaluated ex vivo in two sheep and intra-thoracically implanted in a sheep. These experiments were terminated at 70, 79, and 17 days due to blood leakage through the connector system within the housing. No thrombus formation was observed in any of the retrieved pumps. A modified Model II with a new connector system was subsequently intra-thoracically implanted in a sheep. The sheep survived for 482 days without any sign of thromboembolic complication or hemolysis at a fixed rotational speed of 1700 rpm and an average pump flow rate of 5 L/min. There was no intra-device thrombus formation or infarct in major organs. The Model III system, consisting of an implantable controller and a new MSCP with a reduced input power of 13 W, has been developed and implanted in a chronic sheep model. The MSCP was implanted in the left pleural space and the controller in the abdominal wall. The experiment is still in progress for more than 30 days without any significant complication to date. These animal studies strongly suggest the feasibility of the MSCP for use as long-term circulatory assist.

Identification of a novel periplasmic catalase-peroxidase KatA of Legionella pneumophila.

A gene katA that encodes a novel catalase-peroxidase was cloned from the chromosome of Legionella pneumophila. The nucleotide sequence revealed that KatA was highly homologous to members of the bacterial bifunctional catalase-peroxidase family. In addition, KatA has a N-terminal signal sequence and was considered to be present in the periplasm of the bacterium.

[Evaluation of sleep fragmentation by EEG spectrography in patients with obstructive sleep apnea syndrome].

Sleep fragmentation is considered to have a central role in the pathophysiology of sleep-disordered breathing. However, the evaluation of sleep fragmentation can be very troublesome work. We sought to evaluate sleep fragmentation in 10 patients with obstructive sleep apnea syndrome by utilizing fast-fourier transforms to render their sleep encephalograms as spectrograms (horizontal axis: time, vertical axis: frequency, color: power). Detailed 5-minute spectrograms revealed relatively uniform patterns of change in EEG spectra during and after apnea episodes. In non-REM sleep, delta activity during apnea was followed by alpha and beta activity during resumed breathing. In REM sleep, flat and low-power spectral patterns during apnea were followed by relatively weak alpha and beta activity during resumed breathing. In compressed, 1-hour views of the spectrograms, repeated apnea events appeared as vertical striped patterns of spectral change. These patterns were thought to be representative of sleep fragmentation. The spectrographic display of EEGs may be a very simple and easy method for the evaluation of sleep-fragmentation in sleep disordered breathing.

Recent progress in the development of Terumo implantable left ventricular assist system.

The research group of the Terumo Corporation, the NTN Corporation, and Setsunan University (T. Akamatsu) has been developing an implantable left ventricular assist system (ILVAS) featuring a centrifugal blood pump with a magnetically suspended impeller (MSCP). The impeller of the MSCP is suspended by a magnetic bearing, providing contact-free rotation of the impeller inside the pump housing. Thus the MSCP is expected to provide years of long-term durability. Ex vivo chronic sheep experiments using the extracorporeal model (Model I) demonstrated long-term durability, nonthrombogenicity, and a low hemolysis rate (plasma free Hb <6 mg/dl) for more than 2 years. The prototype implantable model (Model II; 196 ml, 400 g) was evaluated ex vivo in 2 sheep and intrathoracically implanted in a small sheep (45 kg). These experiments were terminated at 70, 79, and 17 days, respectively, because of blood leakage through the connector system within the housing of Model II. There was no thrombus formation on the retrieved pump surfaces. A new connector system was introduced to the Model II pump (modified Model II), and the pump was intrathoracically implanted in a sheep. Pump flow rate was maintained at 3-7 L/min at 1700-1800 rpm. The temperature elevation on the surfaces of the motor and the electromagnet inside the pump casing was kept less than 6 degrees C. The temperature of the tissue adjacent to the pump casing became normal 10 days postoperatively. The sheep survived for more than 5 months without any sign of mechanical failure or thromboembolic complication. In vitro real-time endurance tests of motor bearings made of stainless steel and silicone nitride have been conducted for more than 1 year without any sign of bearing wear. The next prototype system (Model III), with an implantable controller and a new MSCP with reduced input power, has been developed with a view toward a totally implantable LVAS.

[Changes in tracheal and lung sounds before and after resection of adenoid cystic carcinoma of the trachea].

A 79-year-old woman was admitted to our hospital with complaints of wheezing and dyspnea. Adenoid cystic carcinoma was diagnosed from the findings on biopsy specimens obtained by fiberoptic bronchoscopy. The tumor was resected and end-to-end anastomosis was performed. Precision analyses of lung sounds were conducted before and after the operation. During eupnea the tracheal sounds prior to operation contained an accentuated, high-frequency component at about 1,500 Hz. The tracheal sounds included a single monophonic wheeze during deep inspiration and 5 wheezes during forced expiration. By contrast, lung sounds in the chest wall were almost normal both before and after the operation. We concluded that increases in high-frequency components in tracheal sounds and the manifestation of various wheezes can be useful in diagnosing tracheal stenosis and/or tumors.

[Screening of sleep apnea/hypopnea syndrome by home pulse oximetry].

We evaluated the diagnostic accuracy and reproducibility of results obtained by home oximetry for the screening of sleep apnea/hypopnea syndrome. Our subjects were 40 patients who underwent home oximetry (12 patients for 1 night, 28 patients for 2 nights) followed by all-night polysomnography. Their mean age was 50.7 +/- 11.2 years; mean body mass index (BMI), 27.6 +/- 4.4 kg/m2; and mean apnea/hypopnea index (AHI), 36.0 +/- 26.0 hr-1. The data obtained by home pulse oximetry were fed into a personal computer, and utilized to calculate the desaturation cycles per hour (oxygen desaturation index (ODI) of 2-4%) and the percentage of time that SpO2 measured less than 90% (T 90). Polysomnography was used to monitor the number of apnea and hypopnea episodes per hour of sleep (AHI). With sleep apnea/hypopnea syndrome defined as AHI > or = 15, the diagnostic sensitivity and specificity of home pulse oximetry were 73.5% and 83.3%, respectively, when ODI-3 > or = 15 was used as the diagnostic standard. Patients who showed false negative results had a lower mean BMI (25.5 +/- 3.0) than those who showed true positive results (28.8 +/- 4.6). The reproducibility of ODI-3 data obtained at home was very high (r = 0.964, n = 28). In conclusion, home pulse oximetry seems to be a very useful tool for the detection of apnea/hypopnea syndrome, but false negative results should be considered a possibility, especially in patients who are not obese.

More than 1 year continuous operation of a centrifugal pump with a magnetically suspended impeller.

The authors have been developing a centrifugal pump with a magnetically suspended impeller (MSCP) designed for total artificial heart and long-term ventricular support. The MSCP consists of a magnetic bearing, an impeller and housing, and a driving motor. The impeller is suspended by a magnetic bearing, therefore providing contact free rotation of the impeller inside the pump. This study was designed to evaluate long-term durability and nonthrombogenicity of the MSCP in a chronic sheep model. The blood contacting surfaces of the pump and conduits were completely modified by a heparin immobilization technique (Hepaface). The MSCP was placed paracorporeally as a left heart bypass between left ventricle and descending aorta in three adult sheep. Coumadin was given orally to maintain prothrombin time at 15-20 sec. The coagulation and hematologic parameters, including plasma free hemoglobin, were periodically monitored throughout the experiment. Under daily movement in the cage, the pump could produce average flow rates of 3-6 L/min (50-100 ml/kg) at 1,700-2,000 rpm. Although the arterial pulse contour decreased, there was no physiologic deterioration. The axial impeller excursion monitored by a position sensor was < 25 microns. Plasma free hemoglobin level remained at < 5 mg/dl throughout the experiment. There was no increase in the motor current, which indicates no massive thrombus formation around the impeller. One experiment was terminated at 70 days due to Hall sensor dysfunction of the motor. The retrieved pump was entirely free from thrombus formation. There was no detectable thrombus formation inside the pump or the inflow and outflow conduits. Hematologic, renal, and hepatic parameters remained within the normal range throughout the experiment. The other two sheep have survived for more than 395 and 41 days without major complication. These studies demonstrated that the MSCP has significant potential for long-term use.

Cloning and sequencing of the dnaK and grpE genes of Legionella pneumophila.

A 4.4-kb DNA fragment from Legionella pneumophila (Lp) was isolated, which could complement an Escherichia coli (Ec) dnaK ts mutant, HC4102. Nucleotide sequence analysis of the region revealed two complete open reading frames (ORFs) encoding both a predicted DnaK protein of 644 aa and a predicted GrpE protein of 199 aa, and also the 5'-end of the predicted dnaJ gene organized in the order of grpE-dnaK-dnaJ. Consensus heat shock (HS) promoter sequences were identified upstream of the start of both grpE and dnaK transcripts. However, no obvious promoter sequences were detected upstream of dnaJ. The transcription start points of grpE and dnaK were determined by primer extension analysis and the amount of each of the transcripts increased four- to eightfold after HS.

New mechanism to reduce the size of the monopivot magnetic suspension blood pump: direct drive mechanism.

Size reduction of the monopivot magnetic suspension blood pump has been achieved by reducing the size of the magnetic suspension and employing a direct drive mechanism in place of a brushless DC motor and a magnetic coupling. The flow has also been improved using a closed hollow impeller to remove flow obstruction at the inlet and using radial straight vanes to reduce the impeller speed by 30%. Hemolysis testing was conducted for the new models. Results showed that model DD1 presented only a slightly higher level of hemolysis than a regular extracorporeal centrifugal pump.

Cloning and nucleotide sequences of iron and copper-zinc superoxide dismutase genes of Legionella pneumophila and their distribution among Legionella species.

A facultative intracellular parasite Legionella pneumophila has two kinds of superoxide dismutase (SOD), iron-containing superoxide dismutase (Fe-SOD) and copper,zinc-containing one (Cu,Zn-SOD). We cloned both SOD genes of L. pneumophila and determined their DNA sequences. The Fe-SOD gene (sodB), isolated by functional complementation of a SOD-deficient Escherichia coli strain, encoded a protein of 192 amino acids conserving the Fe-SOD-specific amino acid residues. A clone containing entire Cu,Zn-SOD gene (sodC) was constructed by connecting two contiguous DNA fragments; one with a lower part of the gene was obtained by colony hybridization with a probe acquired by polymerase chain reaction (PCR) with degenerate oligonucleotide primers corresponding to conserved regions of known Cu,Zn-SOD genes and the other with an upper part of the gene was by IPCR (inverted PCR). The sodC gene encoded a protein of 162 amino acids, of which the first 20 amino acids inferred a signal peptide similar to other bacterial Cu,Zn-SODs reported previously. Both clones expressed their SOD activities in E. coli K-12 through their own plausible promoters. We examined for SOD genes on chromosomes of several Legionella species. All chromosomes were hybridized with Fe-SOD gene of L. pneumophila, but Cu,Zn-SOD gene did not hybridize to the chromosomes of other than L. pneumophila strains.

[Automated analysis of all-night records of tracheal sound to detect sleep disordered breathing].

In sleep-disordered breathing, tracheal sounds disappear during apnea and vary cyclicly during hypopnea. We tried to detect these changes in tracheal sounds automatically with a personal computer, and we evaluated the diagnostic accuracy of this system for detecting sleep-disordered breathing. Polysomnography and tracheal sound recording were done in 33 subjects with possible sleep apnea/hypopnea syndrome. Eighteen had positive results defined as an apnea/hypopnea index greater than 15. Tracheal sounds were digitized and a personal computer was used to calculate short time power spectra (44-600 Hz) every 0.2 seconds by the fast Fourier transform. The moving averages (18 seconds) of the logarithms of the power spectra were calculated every 2 seconds. Transient falls in the moving averages of more than 12 dB were detected. Those that were lower than 5 dB above the level of background noise were classified as tracheal sound apneas. The number of falls of more than 12 dB and the number of tracheal sound apneas correlated strongly with the number of apneas plus hypopneas (r = 0.95) and with the number of apneas (r = 0.97), respectively. The sensitivity and specificity of tracheal sound analysis for the sleep apnea/hypopnea syndrome (as defined above) were 89% and 60%, respectively, when the criteria was more than 15 falls of more than 12 dB per hour. We conclude that tracheal sound analysis by this method is useful as a screening test for the sleep apnea/hypopnea syndrome.

[Hypopnea according to various definitions and coexisting arousal response in patients with sleep-disordered breathing].

Hypopnea is a type of sleep-disordered breathing, and the apnea plus hypopnea index (AHI) is usually computed to diagnose this condition. Nevertheless, there is no consensus on the definition of hypopnea. To arrive at a reasonable definition, we examined the relationship between arousal response and hypopnea as defined in various ways, in polysomnographs of 20 patients with sleep-disordered breathing. Hypopnea was defined as a 50% reduction in thoraco-abdominal movement of various durations (all of which were more than 10 sec) and coexisting oxygen desaturation of various degree. The ratio of coexisting arousal response for hypopneas was higher when hypopnea was defined as having a relatively long duration and relatively deep desaturation, but more arousal responses were missed. 1) When the threshold duration for hypopnea was fixed at 10 sec, the following results were obtained: With desaturation thresholds of 4% and 2%, the mean ratios of missed arousals were 37% (range, 1-85%) and 12% (range, 0.5-35%), respectively. The hypopneas with only 2% desaturation had coexisting arousal responses with a mean frequency of 49% (range, 27-89%). 2) When desaturation was not included in the definition, the following results were obtained: Hypopneas lasting less than 15 sec had lower frequencies of a coexisting arousal response than did those of longer duration. When the threshold duration for hypopnea was 15 sec, the mean ratio of missed arousal was 16.2% (range, 1.6-32.9%).(ABSTRACT TRUNCATED AT 250 WORDS)

[Relationship between power spectra of breath sounds and inspiratory flows at different frequencies].

Regional ventilation and changes in airway caliber have been studied by analysis of breath sounds. The aim of this study was to find which frequency band is most suitable for such purposes. The subjects were 19 healthy men. Breath sounds at 6 sites on the chest wall, airflow rate at the mouth, and ECG were digitized and recorded for 30 seconds. Power spectra of breath sounds at various frequency bands (10-50 Hz, 50-100 Hz, 100-150 Hz, ...., 950-1000 Hz) were calculated with a fast-Fourier transform (FFT) for every block of 512 points (102.4 msec), and mean airflow rates for the blocks were calculated. Data recorded during inspiration when airflow ranged between 0.5 L/s and 3 L/s were analyzed. For frequency bands from 150 Hz to 850 Hz the logarithms of the power spectra were linearly correlated with the logarithms of the airflow rates, and the correlation coefficients exceeded 0.8, but for lower frequency bands the correlations were poor. When the data contaminated by heart sounds and those recorded late in inspiration were excluded the correlations at frequency bands below 150 Hz improved. The slope of log (power)/log (flow) was about 4 for the bands from 100 to 300 Hz, but it became steeper (4 to 6) for higher frequency bands. This means that the power spectra were proportional to the fourth power of the airflow rate for bands below 300 Hz, but the relation was from the fourth to the sixth power for higher frequency bands.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA binding domains in Tn3 transposase.

Various segments of Tn3 transposase were fused individually to beta-galactosidase, and the resulting fusion proteins were examined for their DNA binding ability by a nitrocellulose filter binding assay. Analyses of a series of the fusion proteins revealed that the N-terminal segment of the transposase (amino acid positions 1-242; the transposase gene encodes 1004 residues in all) had specific DNA binding ability for the 38 bp terminal inverted repeat (IR) sequence, and the central segment (amino acid positions 243-632) had non-specific DNA binding ability. Further analyses of each of the two regions revealed that the N-terminal segment could be divided into at least two subsegments (amino acid positions 1-86 and 87-242), neither of which had specific DNA binding ability, but which both possessed non-specific DNA binding ability. The central segment included two subsegments (amino acid positions 243-289 and 439-505) with non-specific DNA binding ability. These results and other observations suggest that Tn3 transposase has several domains including those responsible for non-specific DNA binding, and a combination of two or more domains gives rise to specific DNA binding activity. The C-terminal segment of the transposase (amino acid positions 633-1004), which is very well conserved among transposases encoded by Tn3 family transposons, had no DNA binding ability. This segment may represent the main part of the catalytic domain responsible for the initiation step of transposition.

The modified buccal musculomucosal flap method for cleft palate surgery.

We have reported previously on a palatoplasty method, called the T-shaped musculomucosal buccal flap method, for the primary repair of a cleft palate. This method has been used on more than 90 patients, and satisfactory outcomes have resulted in terms of maxillar development, the prevention of fistulation, and verbal functions. However, 14.3 percent of these patients exhibited a velopharyngeal incompetence that showed no potential improvement through training. In the majority of these patients, the entire raw surface of the oral cavity side could not be covered with a buccal musculomucosal flap, and as a result, postoperative contraction of the soft palate occurred. Thus a new surgical method has proven effective in which both buccal musculomucosal flaps are used as an oral lining, the nasal mucosa having been extended by Z-plasty. We have performed 25 operations using this new method and have observed no postoperative contractions of the soft palate, notwithstanding two cases (8.0 percent) of postoperative fistulation.

Functional analysis of the two domains in the terminal inverted repeat sequence required for transposition of Tn3.

Bacterial transposon Tn3 has a 38-bp terminal inverted repeat (IR) sequence. The IR sequence has been divided into two domains, A and B, of which domain B is bound by transposase, and domain A is not Here, we defined the two domains more precisely by constructing three IR mutants with a 2-bp substitution at relevant sites within the IR sequence, followed by examination of the binding of transposase to the fragments containing these IR mutants: domain A was located at bp 1-11, whereas domain B was at bp 12-38. To see if the two domains in the IR are functionally distinct, we constructed mini-Tn3 derivatives flanked by two IRs with various 2-bp substitutions within domain A or B, and analyzed their ability to mediate cointegration. The mini-Tn3 derivatives flanked by IR(A+ B+) and IR(A- B+) [or IR(A+ B-)] and those flanked by IR(A-B+) and IR(A+ B-) mediate cointegration more efficiently than the mini-Tn3 derivatives flanked by two IR(A- B+)s or by two IR(A+ B-)s. These results and others presented here indicate that the two domains of IR are functionally distinct in promoting cointegration.