Molecular epidemiology and antimicrobial susceptibility of Clostridium difficile isolated from a university teaching hospital in Japan.

Clostridium difficile infection control strategies require an understanding of its epidemiology. In this study, we analysed the toxin genotypes of 130 non-duplicate clinical isolates of C. difficile from a university hospital in Tokyo, Japan. Multilocus sequence typing (MLST) and eBURST analysis were performed for these isolates and nine strains previously analysed by polymerase chain reaction (PCR) ribotyping. Minimum inhibitory concentrations (MICs) were determined for six antibiotics, and the bacterial resistance mechanisms were investigated. Ninety-five toxigenic strains (73%), including seven tcdA-negative, tcdB-positive and cdtA/cdtB-negative strains (A(-)B(+)CDT(-)) and three A(+)B(+)CDT(+) strains, and 35 (27%) non-toxigenic strains, were classified into 23 and 12 sequence types, respectively. Of these, sequence type (ST)17 (21.8%) was the most predominant. MLST and eBURST analysis showed that 139 strains belonged to seven groups and singletons, and most A(+)B(+)CDT(-) strains (98%, 89/91) were classified into group 1. All isolates were susceptible to metronidazole, vancomycin and meropenem; the ceftriaxone, clindamycin and ciprofloxacin resistance rates were 49, 59 and 99%, respectively. Resistance rates to ceftriaxone and clindamycin were higher in toxigenic strains than in non-toxigenic strains (P < 0.001). All ST17 and ST81 strains were resistant to these antibiotics. The clindamycin- and fluoroquinolone-resistant strains carried erm(B) and mutations in GyrA and/or GyrB, respectively. To our knowledge, this is the first MLST-based study of the molecular epidemiology of toxigenic and non-toxigenic strains in Japan, providing evidence that non-toxigenic and toxigenic strains exhibit high genetic diversity and that toxigenic strains are more likely than non-toxigenic strains to exhibit multidrug resistance.

Telaprevir impairs renal function and increases blood ribavirin concentration during telaprevir/pegylated interferon/ribavirin therapy for chronic hepatitis C.

We aimed to examine the relationship between renal dysfunction and anaemia that may develop during combination therapy involving pegylated interferon, ribavirin and telaprevir (PEG-IFN/RBV/TVR) for the treatment of chronic hepatitis C. Sixty-eight patients with genotype 1b high viral loads were treated with PEG-IFN/RBV/TVR. Peg-IFN and RBV doses were administered according to body weight. TVR was prescribed at 2250 mg/day for 44 patients and at 1500 mg/day for 24 patients who had low haemoglobin level (<12 g/dL). When anaemia had developed, the RBV dose was decreased. The serum TVR concentration at day 8 was measured, and the serum RBV concentration was measured serially. The estimated glomerular filtration rate (eGFR) was estimated to assess renal function. At week 1, serum TVR concentration was not correlated with a decrease in eGFR; however, the TVR dose, on a weight basis (mg/kg), and eGFR were correlated (r = 0.2691; P = 0.0265). Moreover, there was a negative correlation between eGFR and RBV serum concentration (r = −0.3694; P = 0.0025), and the serum RBV concentration and decrease in the haemoglobin were significantly correlated from week 1 to week 8. In triple therapy, the TVR dose per weight is correlated with a decline in renal function. Thus, the serum concentration of RBV increases, with a concomitant decrease in haemoglobin. It is important to adjust the doses of TVR and RBV to avoid excessive serum RBV levels and the development of severe anaemia, to achieve a good clinical effect.

Associations between capsular serotype, multilocus sequence type, and macrolide resistance in Streptococcus agalactiae isolates from Japanese infants with invasive infections.

SUMMARY Streptococcus agalactiae (group B streptococcus; GBS) isolates (n = 150) from infants with invasive infections between 2006 and 2011 were analysed for capsular serotype, multilocus sequence type, and antibiotic susceptibility. In cases with late-onset disease (n = 115), primary meningitis was predominant (62.6%), but represented only 39.1% in cases with early-onset disease (n = 23). The most common serotype was III (58.7%), followed by Ia (21.3%) and Ib (12.7%). Sequence types (STs) of serotype III strains included ST17 (50.0%), ST19 (26.1%), ST335 (18.2%), ST27 (4.5%), and ST1 (1.1%). Predominant STs of serotypes Ia and Ib were ST23 (81.3%) and ST10 (84.2%), respectively. No penicillin-resistant strains were detected, but 22·0% of strains had mef(A/E), erm(A), or erm(B) genes, which mediate macrolide resistance. A new ST335, possessing an mef(A/E) gene belonging to clonal complex 19 gradually increased in frequency. Improved prevention of invasive GBS infections in infants requires timely identification, and ultimately vaccine development.

Development of a long-slot microwave plasma source.

A 20 cm long 10 cm wide microwave plasma source was realized by inserting two 20 cm long 1.5 mm diameter rod antennas into the plasma. Plasma luminous distributions around the antennas were changed by magnetic field arrangement created by permanent magnets attached to the source. The distributions appeared homogeneous in one direction along the antenna when the spacing between the antenna and the source wall was 7.5 mm for the input microwave frequency of 2.45 GHz. Plasma density and temperature at a plane 20 cm downstream from the microwave shield were measured by a Langmuir probe array at 150 W microwave power input. The measured electron density and temperature varied over space from 3.0 × 10(9) cm(-3) to 5.8 × 10(9) cm(-3), and from 1.1 eV to 2.1 eV, respectively.

Dust particle diffusion in ion beam transport region.

Dust particles of µm size produced by a monoplasmatron ion source are observed by a laser light scattering. The scattered light signal from an incident laser at 532 nm wavelength indicates when and where a particle passes through the ion beam transport region. As the result, dusts with the size more than 10 µm are found to be distributed in the center of the ion beam, while dusts with the size less than 10 µm size are distributed along the edge of the ion beam. Floating potential and electron temperature at beam transport region are measured by an electrostatic probe. This observation can be explained by a charge up model of the dust in the plasma boundary region.

3-Isopropylmalate dehydrogenase from chemolithoautotroph Thiobacillus ferrooxidans: DNA sequence, enzyme purification, and characterization.

3-Isopropylmalate dehydrogenase encoded by the Thiobacillus ferrooxidans leuB gene was purified to homogeneity from Escherichia coli cells harboring a recombinant plasmid containing the leuB gene. The native enzyme molecule is a dimer of molecular weight 38,000. The Km value for 3-isopropylmalate was estimated to be 26 microM and that for NAD+ 0.8 mM. The presence of K+ or NH4+ is essential for the enzyme reaction. The enzyme is activated about 4-fold by the addition of 1.0 mM Mg2+ or Co2+. The optimum pH and temperature for the activity are 9.0 and 60 degrees C, respectively. The properties of the enzyme are similar to those of the Salmonella typhimurium and Thermus thermophilus enzymes, except for substrate specificity. T. ferrooxidans 3-isopropylmalate dehydrogenase is able to utilize D- and L-malate as substrates in addition to 3-isopropylmalate. Sequencing of subcloned DNA revealed that the leuB gene consists of a 1,074 bp open reading frame and encodes 358 amino acid residues corresponding to the subunit (38,462 Da). The amino acid sequence of 3-isopropylmalate dehydrogenase from T. ferrooxidans and those of some heterotrophic microorganisms have high homology.

Immunosuppressants and calcineurin inhibitors, cyclosporin A and FK506, reversibly inhibit epileptogenesis in amygdaloid kindled rat.

Calcineurin (CaN) immunoreactivity and content increased markedly in kindled rat brain, and this increment was due to CaN in the membrane fraction. Investigation of the effects of cyclosporin A and FK506 (immunosuppressants which inhibit CaN activity in T lymphocytes) in the kindling phenomena showed that the kindling stage progression was reversibly blocked by these drugs. These findings suggest that calcineurin may play an essential role in acquiring epileptogenesis in kindling.

Evaluation of recombinant human granulocyte colony-stimulating factor (rhG-CSF) therapy in granulopoetic patients complicated with sepsis.

A study was carried out to investigate the efficacy of therapy with recombinant human granulocyte colony-stimulating factor (rhG-CSF) in 24 patients with granulocytopenia and sepsis who had failed to respond to antibiotics. The mean leukocyte count at the start of the study was 911 +/- 334/microliter. Patients were injected subcutaneously with 75 micrograms rhG-CSF once daily for a mean of 5.2 days. The plasma G-CSF concentration was measured by ELISA. The leukocyte count increased approximately 9-fold after 1 week in 19 patients and the percentage of granulocytes rose from 46.2% to 78.9%. These 19 patients survived, while the 5 patients with no leukocyte response to rhG-CSF died. High plasma G-CSF levels were found in patients with granulocytopenia. Plasma G-CSF levels decreased as levels of granulocyte increased in survivors. A high plasma G-CSF concentration persisted in the 5 non-responding patients resulting in a fatal outcome. This study suggests that rhG-CSF both increased the leukocyte count and was a useful therapeutic manoeuvre for sepsis.

Expression of cyclin-dependent kinase 5 and associated cyclins in Leydig and Sertoli cells of the testis.

In this study, we examined the expression and subcellular localization of cyclin-dependent kinase 5 (Cdk5), cyclin D1, and cyclin E in Leydig and Sertoli cell lines that were cultured with 7.5, 1.0, 0.5, or 0% serum (mixture of a 2:1 ratio of horse serum and fetal bovine serum) and in the developing rat testis to verify the possible functions of Cdk5, cyclin D1, and cyclin E in the testis. The abundance of Cdk5 and cyclin E in the Leydig cell line, TM3, was significantly reduced at low serum concentrations. In contrast, serum concentration had no effect on Cdk5 and cyclin E levels in the Sertoli cell line, TM4. Cyclin D1 was detected by western blot analysis in TM4 cells only, and its abundance was serum dose dependent. The kinase activity of Cdk5 in TM3 and TM4 cells that were cultured at various serum concentrations coincided with the levels of Cdk5 expression. Immunohistochemical staining for Cdk5 and cyclin E revealed nuclear and cytoplasmic distribution, both in TM3 and TM4 cells. Moreover, cyclin D1 immunoreactivity was only detected in TM4 cells. In the developing rat testis, Cdk5 expression was most prominent at 2 and 3 weeks after birth. Cyclin D1 was strongly expressed at 1 and 2 weeks in premature rat testes. On the other hand, cyclin E was highly expressed in the adult testis. Immunohistochemical localization of Cdk5, cyclin D1, and cyclin E in 1-week-old and adult rat testes revealed expression in both Leydig and Sertoli cells. Our results suggest that Cdk5 in TM3 and Leydig cells of the testis might play a role in cell cycle regulation, whereas Cdk5 in TM4 and Sertoli cells of the adult testis might have some additional functions besides control of proliferation.

Treatment of metastatic liver tumors by intermittent repetitive injection of an angiogenesis inhibitor using an implantable port system in a rabbit model.

Angiogenesis inhibitors are expected as a new type of anticancer drug, because they may prevent tumor neovascularization. Among several angiogenesis inhibitors, 6-o-(N-chloroacetylcarbamoyl)-fumagillol (TNP-470) is thought to inhibit the proliferation of migrating endothelial cells, as an aspect of angiogenesis, and attracts an attention for clinical application. We attempted to evaluate anticancer effects of intermittent repetitive intraportal injection of TNP-470 for metastatic liver tumor models and considered the usefulness and a role as an angiogenesis inhibitor, TNP-470, in the anti-cancer therapy using an implantable port system. We used 25 Japanese white rabbits with metastatic liver tumors made by intraportal injection of tumor cells (VX2-carcinoma), and divided those into five groups; group A was control group, group B was treated by repetitive subcutaneous injection of TNP-470 and group C, D, E was treated by repetitive intraportal injection of TNP-470, adriamycin, TNP-470 + adriamycin respectively 5 times at 2 or 3 days interval after tumor inoculation using implantable port systems. After 2 weeks, we evaluated anti-cancer effect of TNP-470 pathologically. In the number of grown tumors, there was no difference among five groups statisitically. But the mean tumor size of each group is smaller in order of group E, D, C, B, A and the differences were significant (P < 0.05) except between E and D group. And intratumoral neovascularization tended to be less in group B, C, E than in group A, D pathologically. We consider that intermittent repetitive injection of TNP-470 is more useful than systemic administration for metastatic liver tumors and the administration of angiogenesis inhibitor with other anti-cancer agents via an implantable port system will be one of the most effective therapeutic methods for them.

[A case of rheumatoid pachymeningitis].

Here we present a 53-year-old woman with rheumatoid pachymeningitis. The subject had rheumatoid arthritis (RA) for 15 years. In April, 1996 she began to experience intermittent headaches. In September, her headaches became severe and continuous. In October, she suddenly developed ptosis of the left eye and diplopia. She also started to have dysphagia and she found it increasingly difficult to eat. She was admitted to our hospital on November 1, 1996. Neurological examinations revealed palsies of the left IIIrd, IVth, and VIth, and bilateral IXth, and Xth cranial nerves. Laboratory findings showed leukocytosis, elevated blood sedimentation rate, and positive CRP. Serum RA titer was positive (30x). The cerebrospinal fluid was normal and bacteriological examination was negative. T1-weighted MRI demonstrated hypertrophic cranial dura extending from the falx cerebri to tentorium cerebelli, which was enhanced markedly by Gd-DTPA. The dura adjacent to the cavernous sinus and the clivus were also thickened, which probably caused her cranial polyneuropathies. The dural biopsy showed massive infiltration of the inflammatory cells throughout the dura, proliferation of collagen fibers, and necrotic granuloma with neutrophilic infiltrations. Neither rheumatoid nodules, nor vasculitis were found. Despite the absence of rheumatoid nodules in the dural biopsy, the clinical features, pathologic specimens, and MRI findings of the thickened dura were most consistent with rheumatoid pachymeningitis. Administration of dexamethason ameliorated her headache on the 4th hospital day, and the cranial polyneuropathies completely disappeared on the 35th hospital day. The dural enhancement previously seen on the contrast T1-weighted MRI was diminished. Serum RA titer was also normalized (10x). Rheumatoid pachymeningitis is an extremely rare disease, and only 16 cases were reported in the literatures. Hypertrophic pachymeningitis should be considered as a diagnostic possibility in RA patients who have prolonged headache, and Gd-DTPA MRI is recommended to demonstrate the dural involvement.

[Basic and clinical evaluations of cefmetazole in postoperative wound infections].

The time course of the concentration of cefmetazole (CMZ) in the serum and in skin and intestinal tissues was determined after a single intravenous injection of 2 g of the drug. CMZ moved into them well. Furthermore, 41 patients with postoperative wound infection (superficial in 29 and deep in 12) were treated with CMZ 2-4 g daily. Bacteriological examination of the lesions with simultaneously carried out. As a result, 101 strains of bacteria were isolated and identified. Mixed infection was found in 27 cases (65.9%). Fifteen strains (14.9%) of E. coli, 15 (14.9%) of B. fragilis, 7 (6.9%) of Klebsiella sp. and 7 (6.9%) of Proteus sp., were the main bacteria isolated. Eight cases (19.5%) had mixed infection of E. coli and B. fragilis. The committee (3 members) evaluated CMZ to be effective in 75.6% (31 of 41 cases) and bacteria disappeared in 60.5% (23 of 38 cases). The side effects observed were pyrosis and feeling of gastric malaise in 1 case. The results suggest that CMZ is useful, which exerts an excellent effect on postoperative wound infections.

[A case of apnea during spinal anesthesia after intravenous injection of sedatives].

During spinal anesthesia for appendectomy (sensory level: -Th7), apnea occurred for about 20 minutes by intravenous administration of diazepam. Since the patient showed hyperventilation sign before the incident, this apnea might be caused by low PaCO2 and stopping respiratory stimulation from psychological unrest when diazepam, pentazocine and droperidol were administered. In the postoperative period, this patient showed normal neurologic and psychosomatic functions. In such a case as this, long duration of apnea might damage brain by severe hypoxia. Apnea after hyperventilation during spinal anesthesia as observed in this case could lead to a serious accident.

[Clinical evaluation of dynamic MRI in the re-diagnosis of therapeutic effect and recurrence after transcatheter arterial embolization for hepatocellular carcinoma].

We evaluated re-diagnostic ability of dynamic MRI as compared with T2 weighted image in the diagnosis of primary therapeutic effect and tumor recurrence after transcatheter arterial embolization (TAE) for hepatocellular carcinoma. Thirty-four nodules in 30 patients with hepatocellular carcinoma were estimated based on operative and angiographic findings. According to the time when dynamic MRI was taken, nodules were classified as a short-term observation group consisting of 15 nodules obtained within a month after TAE, or as a long-term observation group consisting of 19 nodules obtained over a month after TAE. In the short-term observation group, sensitivity, specificity, accuracy was 89%, 100%, 93% on dynamic MRI and 78%, 67%, 73% on T2WI, respectively. In the long-term observation group, these were 94%, 100%, 95% on dynamic MRI and 100%, 33%, 89% on T2WI and 94%, 67%, 89% on Lipiodol-CT, respectively. In both groups, dynamic MRI was superior in accuracy. Conclusively, we consider that dynamic MRI is a most accurate diagnostic method that should be added to routine MRI after TAE. Especially in the case diagnosed positive on T2WI, the usefulness of dynamic MRI should be emphasized to determine the schedule of the therapy, because the cases that were false positive on T2WI were accurately diagnosed on dynamic MRI.

[Useful treatment modality for postoperative duodenal fistula with somatostatin analogue].

A case with leakage of duodenal stump treated by a somatostatin analogue (SMS 201-995,SMS) effectively was reported. A 39-year-old man had underwent gastric resection (Billroth I) and distal pancreaticojejunostomy for transsection of duodenal bulbus and pancreatic body. After 14 days later duodenal diverticulization was performed due to anastomotic stricture of the gastro-duodenostomy, which resulted in leakage of duodenal stump and voluminous fluids from drains were lasting about 5 weeks. Amylase (AMY) value was shown as high as 703,812 U/l in the exudate. SMS was injected by 100 micrograms, s, c, by three times a day for 24 days. The volume of exudate decreased immediately after the administration of SMS and to one-half in volume in 2 weeks. AMY also fell to 42, 378 U/l. The drain was removed two months after the third surgery. The patient was discharged on post operative day 200. SMS was known to inhibit secretion of bile and intestinal as well as pancreatic juice and its usefulness for leakage of the intestinal tract, especially of the duodenum, is suggested.

[A case of spontaneous rupture of the stomach].

We experienced a case of rupture of the stomach which was a 80-year-old female, and had suffered from vomiting after lunch followed by abdominal pain and distention. Physical examination revealed muscle guarding and radiography depicted massive free gas in the abdominal cavity. At laparotomy, a rupture was located close by the anterior wall of the greater curvature of the stomach. There was no evidence of ulceration, tumorigenesis, or traumatic history. The operation consisted of debridement around the rupture site and subsequent closure. The rupture was considered to have occurred at a fragile site due to circulatory disturbance of the gastric wall.

[DS-bronchial arteriography; demonstration of metastasized lymphnodes of lung cancer--with special reference to the primary effect in BAI].

We analysed the 44 DS-Bronchial arteriograms (DSBAG) of lung cancer with an attention to the demonstration of metastasized mediastinal lymphnodes. The stain of lymphnode is well demonstrated in the pericarinal region, such as station #7, #R4 along the course of bronchial arteries. The swollen nodes with stains on DSBAG show the good reduction rates (37.5%) after BAI that is analogous to the primary lesion (38.9%), in contrast to the nodes without stains (24.5%). DSBAG with excellent contrast resolution contributes to the BAI as verifying the infused area.

[High resolution computed tomography of diffuse interstitial pneumonia during treatment].

HRCT was carried out in twenty patients with diffuse interstitial pneumonia: 13 cases of IIP, 3 of BOOP, 2 of drug-induced pneumonia, 1 of rheumatoid lung and acute interstitial pneumonia of unknown origin. With special attention to inflammatory activity, the patients underwent HRCT periodically during the treatment. Correlative investigation between HRCT image and grade of accumulation in 67Ga scintigraphy was also performed. Response to steroid therapy was clearly reflected on HRCT image, that was shown as decreasing pulmonary density or thinning of honeycomb wall. HRCT is considered to be useful in assessing the activity of diffuse interstitial pneumonia.

[MR imaging for lung cancer in evaluating local invasion; referring to CT and surgical-histological basis].

The diagnostic potentiality of MR imaging for lung cancer was researched in 17 patients in evaluating local invasions based on surgical-histological evidence. Comparative reviewing with CT was also performed. MR imaging appeared to have an ability unique to portray malignant invasions somewhere such as in the hili or apices. On the other hand, there were also some diagnostic limitations of MR imaging in delineating the localized invasive changes. On the way of working up lung cancer, CT still may stand at the first choice as a radiographic staging procedure which provides the more detailed anatomic-pathologic informations. We consider that MR imaging is mandatory when CT and/or other modalities could'not obtain efficient results.

[CT of hypersensitivity pneumonitis].

High resolution computed tomography (CT) was applied to seven patients with hypersensitivity pneumonitis clinically confirmed. The CT findings include; (1) granular pattern with acino-centric distribution, (2) increase in density of haziness in the lung fields, (3) multiform high density areas, and (4) "subpleural curvilinear shadow" localized posteriorly. Of these findings, which severally varied degree during clinical course, the haziness showed the most remarkable fluctuation. It was especially of interest that the relapsed haziness distributed quite in the same regions. High resolution CT may be an effective adjuvant tool for hypersensitivity pneumonitis when applied to the opportune evaluation through the clinical course.