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A novel Gram-negative, aerobic, rod-shaped, non-spore-forming bacterial strain, RYU5 T , was isolated from a stool sample of an inpatient at a hospital in Okinawa, Japan. The optimal growth temperature of RYU5 T was 30 °C. Phylogenetic analysis based on the sequences of housekeeping genes, including the 16S rRNA, rpoB, rpoD and gyrB genes, showed that RYU5 T was a member of the Pseudomonas putida group and was located close to Pseudomonas monteilii and P. putida. Whole-genome comparisons, using average nucleotide identity and digital DNA-DNA hybridization, confirmed that strain RYU5 T should be classified as a novel species of Pseudomonas. Phenotypic characterization tests showed that utilization of d-mannose, d-serine, l-arabinose and d-fructose could distinguish this strain from other related species of the genus Pseudomonas. Based on genetic and phenotypic evidence, strain RYU5 T should be classified as a novel species, for which the name Pseudomonas asiatica sp. nov. is proposed. The type strain is RYU5 T (=DSM 107182 T , =JCM 32716 T ), with a DNA G+C content of 62.25 mol%.

A carbapenem-resistant and colistin-heteroresistant clinical isolate of Enterobacter cloacae was obtained from an inpatient in Okinawa, Japan. The minimum inhibitory concentrations of both imipenem and meropenem were 32 μg/mL. The isolate showed heteroresistance to colistin using the Etest method and resistance to colistin using the broth microdilution method. It had a disrupted ompC and a mutation in the promoter region of bla ACT-2 , but did not harbor any genes encoding carbapenemase. The disruption of ompC and the mutation in bla ACT-2 was associated with the carbapenem resistance of this isolate. This isolate also had mutations in pmrAB and phoPQ encoding two-component regulatory systems, which may be associated with colistin heteroresistance., (Copyright © 2018. Published by Elsevier Ltd.)

The modified carbapenem inactivation method (mCIM) is a simple phenotypic screening method for detecting carbapenemase production by Enterobacteriaceae and Pseudomonas aeruginosa. We recently developed another modified carbapenem inactivation method (CIMTris), in which carbapenemase is extracted from bacteria with Tris-HCl buffer, to detect carbapenemase production by Acinetobacter and Pseudomonas species. This study describes an improved carbapenem inactivation method, CIMTrisII, for detecting carbapenemase production by Gram-negative pathogens, including Enterobacteriaceae, Acinetobacter and Pseudomonas species. CIMTrisII was different from CIMTris in the concentration of Meropenem disks (5-µg MEM disks vs. 10-µg MEM disks), the inoculum volume of the bacteria (a 5-µl loopful vs. a 10 µl loopful) and the incubation time (1 vs. 2 h). CIMTrisII showed an overall sensitivity of 99.3 % and an overall specificity of 95.0 % for tested isolates. In comparison, CIMTris showed a sensitivity of 96.1 % and a specificity of 96.3 %, and mCIM showed a sensitivity of 67.1 % and a specificity of 100 %. CIMTrisII is thus deemed useful for detecting carbapenemase production by Gram-negative pathogens.

Several species of Aeromonas produce the enzyme CphA metallo-β-lactamase. This study describes an isolate of Aeromonas hydrophila harbouring an acquired gene encoding the carbapenemase GES-24. This isolate was obtained from an inpatient in Okinawa, Japan, with no apparent record of travelling overseas. The minimum inhibitory concentrations of carbapenems against this isolate were 8 µg ml -1 for imipenem and 16 µg ml -1 for meropenem. Recombinant GES-24 hydrolyzed all of the tested β-lactams, including imipenem and meropenem. The genomic environment surrounding blaGES-24 was intI1-blaGES-24-aac(6')-IIc-qacEdelta1-sulI-orfX-tetR-tetE. This is the first report of A. hydrophila producing a GES-type carbapenemase.

The mcr-1 gene encodes a phosphoethanolamine transferase that confers resistance to colistin by transferring phosphoethanolamine to lipid A. A 33-kb IncX4 plasmid harboring mcr-1 was detected in a Klebsiella pneumoniae isolate and two Escherichia coli isolates, and a 66-kb IncI2 plasmid was detected in three E. coli isolates in hospitals in Okinawa, Japan., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

This study describes highly aminoglycoside-resistant Klebsiella pneumoniae and Klebsiella oxytoca clinical isolates obtained from an inpatient in Okinawa, Japan, with no known record of traveling overseas. The minimum inhibitory concentrations of amikacin and arbekacin against these strains were >1024 μg/ml. Whole-genome sequencing analysis revealed that these isolates harbored armA, which encodes a 16S rRNA methylase, ArmA, that confers pan-aminoglycoside resistance. This is the second report of K. pneumoniae harboring armA and the first report of K. oxytoca harboring a 16S rRNA methylase encoding gene in Japan., (Copyright © 2017 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Small-colony variants (SCVs) were obtained from an Enterobacter cloacae clinical isolate in Okinawa, Japan. One variant showed auxotrophy for hemin with a deletion of 20 365 nucleotides, dosC-ydiK-mmuP-mmuM-tauA-tauB-tauC-tauD-hemB-yaiT-yaiV-ampH-yddQ-sbmA-yaiW-yaiY-yaiZ, including hemB, and was more resistant to aminoglycosides and carbapenems, but more susceptible to aztreonam, than the parent strain.

The carbapenem inactivation method (CIM) and modified CIM (mCIM) are simple and economical phenotypic screening methods for detecting carbapenemase production in Gram-negative bacteria. Although the mCIM has been recommended by the Clinical and Laboratory Standards Institute, both the CIM and mCIM have limitations. This study describes another modified CIM, called CIMTris, in which carbapenemase was extracted from bacteria with 0.5 M Tris-HCl (pH 7.6) buffer. The ability of the CIMTris to detect carbapenemase production was examined in Acinetobacter and Pseudomonas species. The CIMTris had an overall sensitivity of 97.6% and an overall specificity of 92.6%, whereas the mCIM had a sensitivity of 45.1% and a specificity of 100% for the isolates tested. These findings indicate that the CIMTris is useful for detecting carbapenemase production in Acinetobacter and Pseudomonas species., (Copyright © 2017 American Society for Microbiology.)

The mcr-1 is a gene encoding a phosphoethanolamine transferase, which confers resistance to colistin by transferring phosphoethanolamine to lipid A. We describe here the emergence of a colistin-resistant Escherichia coli clinical isolate harboring plasmid-mediated mcr-1 in Japan. The isolate belonged to ST5702 and is suspected to come from livestock and transmitted to human. This is the first report of a clinical isolate harboring mcr-1 in Japan., (Copyright © 2017. Published by Elsevier Ltd.)

It is well known that some isolates of Staphylococcus aureus produce pathogenic toxin, Panton-Valentine leukocidin (PVL), and that the toxin has been reported to be highly associated with community acquired methicillin resistant S. aureus (CA-MRSA). Currently, the PCR method using specific primers for the PVL gene (LukS-PV-lukF-PV) have been widely used to detect PVL. In this study, we evaluated the PVL-RPLA "Seiken", diagnostic reagent based on a reserved passive latex agglutination reaction with a specific monoclonal antibody for detecting PVL. A total of 630 clinical isolates were used. PCR method detected 34 PVL-positive (28 MRSA and 6 MSSA), and, of these, PVL-RPLA "Seiken" read positive for 32 isolates (27 MRSA and 5 MSSA), the result indicating two false negative occurrences. The concordance rate was 99.7%. In addition the recommended BHI broth, CCY medium, Dolman broth and Todd-Hewitt broth were applied for toxin preparation media. Toxin concentration produced in CCY medium was significantly higher than those in the remaining culture medium (p < 0.05). PVL-RPLA "Seiken" is a method for detecting the PVL in the culture broth by antigen antibody reaction after an overnight shaking culture. This method does not require any expensive equipments or facilities. Thus this reagent provides us with rapid, easy-to-perform, less expensive test method to detect PVL in clinical microbiology laboratories.

We are making efforts to reduce the number of venipuncture tubes for blood-based testing. On the reconstruction of hematology system in 2011, we planned the system to include hemoglobin A1c (HbA1c) assay and to replace the assay instrument for erythrocyte sedimentation rate (ESR) to use EDTA-2K based whole blood. Accordingly, the revised system required a single test tube for hematological testing, resulting in reduction of blood volume collected. It was estimated that the whole blood collected from outpatients in a year decreased from 143 L to 109 L. Also, the times required to complete venipuncture after outpatient accession were significantly shortened to 10(0.71 +/- 0.27) (2.75-9.55) min, and nearly 50% of outpatients experienced < 2 min of waiting. As the times required for venipuncture were shortened, the turnaround times (TATs) from outpatient accession to finally reporting the test results to physicians were also shortened in the blood-based laboratories. The TATs after outpatient accession to reporting the test results in biochemistry and serology ranged 59 to 80 min (90%-tile), indicating 8 to 16 min less when compared with those before system reconstruction. In conclusion, the decrease in number of venipuncture tubes in hematological testing enables us to reduce the blood volume collected, and to shorten (1) times required for venipuncture procedure, (2) waiting times, and (3) TATs for blood-based testing. However, as demonstrated in HbA1c, i.e., a 50%-tile of TAT for HbA1c delayed for 5 min, the configuration of assay system can greatly influence the TATs of individual test parameters.

Panton-Valentine leukocidin (PVL) is a pore-forming cytotoxin that is produced by Staphylococcus aureus closely associated with skin and soft-tissue infections (SSTI). PVL-positive S. aureus strains have been identified worldwide, including in the USA; however, few studies have reported the presence of these strains in Japan. In this study, we prospectively investigated the prevalence of PVL in S. aureus strains from outpatients presenting with SSTI in Okinawa and characterized the PVL-positive S. aureus strains by polymerase chain reaction (PCR) and multilocus sequence typing (MLST). From 2008-2010, 499 clinical samples were obtained from 497 people. S. aureus was identified in 274 samples, and 36% (99 of 274) were methicillin-resistant S. aureus (MRSA). Seventeen (6.2%) PVL-positive S. aureus strains were detected by PCR, and 12 of the 17 PVL-positive strains were MRSA. Most PVL-positive S. aureus caused furuncles or carbuncles. Nine of the 17 PVL-positive isolates had an ST8 MRSA genotype and most harbored SCCmec type IVa and the arcA gene of the arginine catabolic mobile element, which is identical to the USA300 clone prevalent in the USA. PVL-positive S. aureus strains were more likely to be resistant to erythromycin (65%) and levofloxacin (53%). PVL-positive S. aureus strains have emerged and are spreading as a causative pathogen for SSTI in Okinawa., (© 2012 Japanese Dermatological Association.)

In response to the revision of social medical insurance policy, in which hospital clinics can additionally charge for laboratory testing when the test results are presented to an outpatient in a print-out form on a visiting day, we evaluated laboratory-spending times, so-called turnaround times (TATs). A total of 14,802 outpatients during the period from October 2010 to May 2011 were enrolled. TATs from venipuncture accession to completing blood collection revealed a log-normal distribution with 5 to 6 min of mode and 10(0.95 +/- 0.26) (4.90 to 16.2) min of mean +/- standard deviation. Order waiting time figured a half-normal distribution, 50% tile and 90%-tile being 4 and 16 min, respectively. TATs of blood collection and order waiting time were significantly influenced by days of the week and accession time. Through analysis of TATs from specimen receipt to reporting test results, it became apparent that the tests determined by immunoassay and erythrocyte sedimentation rate (ESR) required more minutes when compared to the remaining tests. Total TATs from venipuncture accession to reporting test results ranged 28 to 29 min (50%-tile) for complete blood count and hemoglobin A1c, whereas those of endocrinology and tumor markers were 65 to 73 min. In conclusion, the tests determined by immunoassay are rate-limiting for rapid reporting efforts in clinical laboratories. Secondly, TATs of blood collection are mostly influenced by order waiting time depending on days of the week and accession time. At present, there is no target value for TATs, however it is important to recognize the necessity to shorten laboratory-spending TATs.

The activity of antibacterial agents against aerobic Gram-positive cocci (25 genus or species, 1029 strains) and anaerobic bacteria (21 genus or species, 187 strains) isolated from clinical specimens in 2008 at 16 clinical facilities in Japan were studied using either broth microdilution or agar dilution method. The ratio of methicillin-resistant strains among Staphylococcus aureus and Staphylococcus epidermidis was 59.6% and 81.2%, suggesting that resistant strains were isolated at high frequency. Vancomycin (VCM), linezolid (LZD) and quinupristin/dalfopristin (QPR/DPR) had good antibacterial activity against methicillin-resistant S. aureus and methicillin-resistant S. epidermidis, with MIC90s of < or = 2 microg/mL. The ratio of penicillin (PC) intermediate and resistant strains classified by mutations of PC-binding proteins among Streptococcus pneumoniae was 92.0% that was highest among our previous reports. Cefpirome, carbapenems, VCM, teicoplanin (TEIC), LZD and QPR/DPR had MIC90s of < or = 1 microg/mL against PC-intermediate and resistant S. pneumoniae strains. Against all strains of Enterococcus faecalis and Enterococcus faecium, the MICs of VCM and TEIC were under 2 microg/mL, and no resistant strain was detected, suggesting that these agents had excellent activities against these species. 15.9% of E. faecalis strains and 1.2% of E. faecium strains showed intermediate to LZD. 17.1% of E. faecium strains showed intermediate or resistant to QPR/DPR. Against all strains of Clostridium difficile, the MIC of VCM was under 1 microg/mL, suggesting that VCM had excellent activity. Carbapenems showed good activity against Clostridiales, Bacteroides spp., and Prevotella spp., but one strain of Bacteroides fragilis showed resistant to carbapenems. And so, the susceptibility of this species should be well-focused in the future at detecting continuously.

We determined MICs of antibacterial agents against 1145 clinical strains of aerobic Gram-negative bacteria (22 species) isolated at 16 Japanese facilities in 2008. MICs were determined using mostly broth microdilution method and antibacterial activity was assessed. Strains producing extended-spectrum beta-lactamases (ESBL) accounted for 3.8% of Escherichia coli, 2.6% of Klebsiella pneumoniae, 6.8% of Klebsiella oxytoca, 5.5% of Proteus mirabilis and 1.8% of Proteus vulgaris. ESBL produced strains were 6.8% at K. oxytoca that increased compared with 3.2% and 5.5% at P. mirabilis that decreased compared with 18.8% in 2006. Among Haemophilus influenzae, 61.7% that decreased compared with 67.7% in 2006, equaled 58.7% in 2004, were strains when classified by penicillin-binding protein 3 mutation. Against Pseudomonas aeruginosa, the activity of most antibacterial agents was similar to that in 2006. Although two antibacterial agents that tobramycin showed an MIC90 of 1 microg/mL and doripenem showed an MIC90 of 4 microg/mL against P. aeruginosa have potent activity. Of all P. aeruginosa strains, 4.3% were resistant to six agents of nine antipseudomonal agents, that decreased compared to 12.2% in 2004 and 5.7% in 2006. Against other glucose-non-fermentative Gram-negative rods, the activity of most antibacterial agents was similar to that in 2006.

USA300 methicillin-resistant Staphylococcus aureus (MRSA) has been attracting worldwide attention as a cause of community-associated MRSA (CA-MRSA) infections in the 21st century. Nosocomial outbreaks of CA-MRSA clones have been progressively more reported in Europe and the USA, but only one very recent report from Kyoto found in Japan. In February 2008, a severe MRSA infection occurred in one immunocompromised patient and three healthy medical staff members at the Department of Dermatology, Graduate School of Medicine, University of the Ryukyus. The epidemiological and clinical pattern of the infection prompted us to characterize the molecular features of the MRSA strain involved. The causative MRSA strain belonged to the multi-locus sequence type 8, staphylococcal cassette chromosome mec (SCCmec) type IVa, spa1 (alternatively t008), agr1 and coagulase type III, and carried the Panton-Valentine leukocidin (PVL) gene and the arginine catabolic mobile element. Pulsed-field gel electrophoresis analysis showed that the MRSA responsible for the outbreak was the USA300 clone. All of the isolated USA300 clones had multiple resistance against six non-β-lactam antimicrobial drugs. We report here the first nosocomial outbreak of multidrug-resistant USA300 MRSA infections in Japan. This report shows that the USA300 clone can manifest severe skin infections such as furuncles and carbuncles even in healthy persons, which require drainage and i.v. treatment, and suggests that the clone can spread in hospital settings worldwide., (© 2011 Japanese Dermatological Association.)

The prevalence of fluoroquinolone-resistant Streptococcus pneumoniae is increasing worldwide. In the present study, a comparison of drug sensitivity and genotypes of clinically isolated strains of levofloxacin (LVFX)-resistant S. pneumoniae obtained from Hong Kong, Okinawa Island and the Japanese main island (Honshu) was performed. MICs of quinolones (LVFX, tosufloxacin, ciprofloxacin, gatifloxacin and sitafloxacin (STFX)) and other antibiotics (penicillin G, cefcapene, cefditoren, clarithromycin and azithromycin) were determined by a microdilution broth method according to the Clinical and Laboratory Standards Institute Standards. The quinolone-resistance determining regions (QRDRs) of gyrA, gyrB, parC and parE of these strains were analyzed by PCR-based sequencing. All 40 strains tested had more than one amino-acid substitution in the QRDRs of gyrA, gyrB, parC or parE. Although there seemed to be some clonality in strains obtained from Hong Kong, there was no clonality in strains obtained from Okinawa and Japan. Strains obtained from Hong Kong, Okinawa Island and the Japanese main island were genetically different by pulsed-field gel electrophoresis analysis. The range of MIC values of STFX against isolates resistant to LVFX (MIC 4-32 mg l(-1)) was 0.12-0.5 mg l(-1), and MIC(80) values of STFX against LVFX-resistant isolates were 0.25 mg l(-1). This study suggests that LVFX-resistant S. pneumoniae is similar in all three locations and STFX is potent against LVFX-resistant S. pneumoniae with multiple mutations in QRDRs of gyrase A and topoisomerase IV.

We determined MICs of antibacterial agents against 1280 clinical strains of aerobic Gram-negative bacteria (19 genus or species) isolated at 16 Japanese facilities in 2006. MICs were determined using mostly broth microdilution method and antibacterial activity was assessed. Strains producing extended-spectrum beta-lactamases (ESBL) accounted for 3.7% of Escherichia coli, 2.7% of Klebsiella spp., and 11.4% of Proteus spp. Notably, 18.8% of Proteus mirabilis was found to produce ESBL higher than 16.7% in 2004. This result was higher extremely than other species. Among Haemophilus influenzae, only 1.2% produced beta-lactamase and 62.8% that increased compared with 57.7% in 2004, were beta-lactamase-negative ampicillin-resistant strains when classified by penicillin-binding protein 3 mutation. Although few antibacterial agents against Pseudomonas aeruginosa have potent activity, only three agents--doripenem, ciprofloxacin, and tobramycin-showed an MIC90 of 4 microg/mL. Of all P aeruginosa strains, 5.7% were resistant to six or more agents of nine antipseudomonal agents, a decrease compared to 8.7% in 2004. Against other glucose-non-fermentative Gram-negative bacteria, the activity of most antibacterial agents was similar to that in 2004.

The activity of antibacterial agents against aerobic Gram-positive cocci (26 species, 1022 strains) and anaerobic bacteria (23 species, 184 strains) isolated from clinical specimens in 2006 at 16 clinical facilities in Japan were studied using either broth microdilution or agar dilution method. The ratio of methicillin-resistant strains among Staphylococcus aureus and Staphylococcus epidermidis was 53.0% and 65.8%, suggesting that resistant strains were isolated at high frequency. Vancomycin (VCM) and quinupristin/dalfopristin (QPR/DPR) had good antibacterial activity against methicillin-resistant S. aureus and methicillin-resistant S. epidermidis, with MIC90s of < or = 2 micrcog/mL. The ratio of penicillin (PC) intermediate and resistant strains classified by mutations of PC-binding proteins among Streptococcus pneumoniae was 87.6%. Ceftriaxone, cefpirome, cefepime, carbapenem antibiotics, VCM, teicoplanin, linezolid(LZD) and QPR/DPR had MIC90s of < or = 1 microg/mL against PC-intermediate and resistant S. pneumoniae strains. Against all strains of Enterococcus faecalis and Enterococcus faecium, the MICs of VCM and TEIC were under 2 microg/mL, and no resistant strain was detected, suggesting that these agents had excellent activities against these species. 10.9% of E. faecalis strains or 3.5% of E. faecium strains showed intermediate or resistant to LZD. 24.4% of E. faecium strains showed intermediate or resistant to QPR/DPR. Against all strains of Clostridium difficile, the MIC of VCM were under 1 microg/mL, suggesting that VCM had excellent activity against C. difficile. Carbapenems showed good activity against Peptococcaceae, Bacteroides spp., and Prevotella spp. However since several strains of Bacteroides fragilis showed resistant to carbapenems and the susceptibility of this species should be well-focused in the future.

Currently, beta-lactamase-negative (BLN) ampicillin-resistant (AR) strains of Haemophilus influenzae are prevalent in Japan. BLNAR strains are defined by the presence of specific mutation(s) in the ftsI gene but are not phenotypically distinguishable by ampicillin (ABPC) susceptibility. In the present study, we showed that cephalexin (CEX), cefsulodin (CFS), and cefaclor (CCL) disk diffusion tests can be effectively used to identify BLNAR strains. A total of 169 clinical isolates of BLN H. influenzae, consisting of 113 of BLNAR and 56 of BLN, ampicillin-susceptible (AS), were included. All the isolates were genetically defined by detection of the TEM gene and partial sequencing of the ftsI gene. The Clinical and Laboratory Standards Institute (CLSI) standard broth microdilution and disk diffusion tests for ABPC provided 20% and 19% false susceptible rates, respectively. Alternatively, 34 cephem agents were tested using disk diffusion. Of the agents tested, CEX, CFS, and CCL disks could effectively discriminate between BLNAR and BLNAS isolates. All the BLNAS isolates showed visible growth inhibitory zones around CEX and CFS disks, but 108 (95.6%) and 106 (93.8%) BLNAR isolates did not. The results indicated 100% predictive values (PVs) for BLNAR and PVs for BLNAS were 91.8% for CEX and 88.9% for CFS. The CLSI-based interpretations for CCL (> or =20 mm) also highly correlated with BLNAR and BLNAS, PVs for BLNAR and for BLNAS being 100% and 93.3%, respectively. With simplicity and discriminability of the test method, we recommend a CEX disk diffusion test in combination with a rapid beta-lactamase test to identify BLNAR isolates in clinical laboratories.

We experienced hospital-acquired infection in March 2008 that three nurses became infected with Panton-Valentine leukocidin (PVL)-positive methicillin-resistant Staphylococcus aureus (MRSA). Accordingly, we performed the retrospective study to determine the prevalence of PVL-positive S. aureus in Okinawa. A total of 731 clinical isolates, consisting of 600 MRSA and 131 methicillin-susceptible isolates in Okinawa, were included. Of the isolates, 16 were positive for PVL gene (lukS-PV-lukF-PV). All the PVL-positive isolates were MRSA, and the first appeared in March 2008. The isolates from the University Hospital were characterized as staphylococcal chromosomal cassette mec type IVa. Through the analysis of pulsed-field gel electrophoresis (PFGE), 16 PVL-positive MRSA isolates were divided in three groups. One isolate (the first group) from the other hospital was less similar (< 40% similarity) when compared with the remaining 15 isolates from the University Hospital. The second group consisted of two respective paired isolates from the same department wards, and those were very similar with each other, indicating possible patient-to-patient transmission. The 11 isolates were characterized as the third group with >80% similarity. The DiversiLab system (bioMérieux) based on repetitive-sequence-based PCR typing demonstrated that the isolates of the third group were similar and indistinguishable with the strains of USA300 clone. However, the first and second groups were not determinable which USA clone was the origin. With these, we could conclude that the PVL-positive MRSA close to USA300 clone first appeared in Okinawa in 2008 and is now becoming prevalent multi-focally. Also, person-to-person transmission is already likely in a hospital setting.

To establish an alternative and more sensitive test method to detect oocyst of Cryptosporidium parvum and cyst of Giardia lamblia in clinical stool specimens, loop-mediated isothermal amplification (LAMP) was evaluated. Minimum cell concentrations at which LAMP assay could detect C. parvum oocyst and G. lamblia cyst were determined as 6.25 x 10(-1) and 3.12 x 10(-1) cells/assay when the stool specimens were spiked with the respective parasites. The results indicated 400 times higher sensitivities or more when compared to the microscopic readings. Twenty and nineteen diarrhea stool specimens spiked with C. parvum oocyst or G. lamblia cyst were assayed by LAMP. The results indicated that 14 (70%) and 16 (84%) samples successfully resulted in positive readings. But the remaining 6 and 3 samples were read as negative probably due to residual stool color. However, further dilutions of DNA extraction samples and addition of bovine serum albumin to LAMP reaction mixture showed positive effects on the occurrence of false-negative readings. With these results, we can conclude that the LAMP assay provides us an accurate and highly sensitive test method to detect C. parvum oocyst and cyst of G. lamblia, in place of labor-intensive and experience-dependent microscopic examination, in clinical laboratories.

Objective: To investigate oral candidiasis in patients with head and neck cancer before, during, and after radiation therapy, and to explore its association with clinical oropharyngeal symptoms., Study Design: A cohort study., Setting: University hospital., Subjects and Methods: Subjects who received radiation therapy (RT) for the treatment of head and neck cancer were divided into two groups: an oral cavity irradiated group (OIRR group, n = 29) and an oral cavity nonirradiated group (ONIRR group, n = 17). A control group consisted of 18 healthy subjects. Patients were examined for signs of oral candidiasis before, during, immediately after, and one month after RT. Mouth and throat soreness (MTS), dysphagia, and xerostomia were evaluated by self-reported questionnaires, and associations between oral candidiasis and these symptoms were analyzed., Results: The incidence of oral candidiasis during RT was significantly higher in the OIRR group (55.2%) than in the ONIRR group (11.8%). Similarly, the occurrence of xerostomia during RT was significantly higher in the OIRR group (86.2%) than in the ONIRR group (52.9%). In the OIRR group, the mean MTS score at the 20th fraction of RT was significantly higher in patients with candidiasis (mean +/- SD, 5.8 +/- 2.1) than in those with RT-induced mucositis without candidiasis (3.7 +/- 2.0). In the OIRR group, 65.2 percent of patients who experienced dysphagia developed oral candidiasis, compared with only 10 percent in the ONIRR group., Conclusion: Oral candidiasis concurrent with oral mucositis due to RT may increase oropharyngeal discomfort during RT., (Copyright (c) 2010 American Academy of Otolaryngology-Head and Neck Surgery Foundation. Published by Mosby, Inc. All rights reserved.)

The publications in 1990s have indicated decreased recovery rates of obligate anaerobes from blood cultures and have questioned the need for routine anaerobic blood culture bottles. In this study, we compared positivities of the paired aerobic and anaerobic bottles and rapidity to detect positive cultures by two automated blood culture systems, BACTEC 9120 and BacT/ALERT 3D. Of 401 positive readings by BACTEC 9120, 338(84.3%) aerobic bottles became to be positive, and anaerobic bottles were 318(79.3%). Also, of 437 positive readings by BacT/ALERT 3D, positivities were 90.8% and 67.3% by aerobic and anaerobic bottles, respectively. These results indicated 5.0% and 23.7% more organisms were recovered in aerobic bottles than in anaerobic bottles, including more staphylococci, gram-positive rods, glucose-nonfermentative gram-negative rods and yeasts. Only 4 (0.14%) of 2,799 BACTEC 9120 anaerobic bottles and 2 (0.06%) of 3,428 BacT/ALERT 3D anaerobic bottles recovered obligate anaerobes. We compared time to detect positive cultures during incubation cycle by both aerobic and anaerobic bottles. Aerobic bottles in BACTEC 9120 read more positive cultures >2 hours earlier than anaerobic bottles, whereas BacT/ALERT 3D could not demonstrate a statistical significance in rapid reading of positive cultures. These results support that recovery rates of obligate anaerobes markedly decreased and that the routine use of anaerobic blood culture bottles is not legitimate at this time. In place of anaerobes, it is an urgent and important issue how to recover fungi correctly and rapidly from blood cultures.

We have reported in this journal in vitro susceptibilities of clinical isolates to antibiotics every year since 1992. In this paper, we report the results of an analysis of in vitro susceptibilities of 12,919 clinical isolates from 72 centers in Japan to selected antibiotics in 2007 compared with the results from previous years. The common respiratory pathogens, Streptococcus pyogenes, Streptococcus pneumoniae, Moraxella catarrhalis and Haemophilus influenzae maintained a high susceptibility to fluoroquinolones (FQs). The resistance of S. pyogenes to macrolides has been increasing every year and this was especially clear this year. Most strains of Enterobacteriaceae except for Escherichia coli showed a high susceptibility to FQs. Almost 30% of E. coli strains were resistant to FQs and the resistance increased further this year. FQs resistance of methicillin-resistant Staphylococcus aureus (MRSA) was approximately 95% with the exception of 45% for sitafloxacin (STFX). FQs resistance of methicillin-susceptible S. aureus (MSSA) was low at about 10%. FQs resistance of methicillin-resistant coagulase negative Staphylococci (MRCNS) was higher than that of methicillin-susceptible coagulase negative Staphylococci (MSCNS), but it was lower than that of MRSA. However, FQs resistance of MSCNS was higher than that of MSSA. FQs resistance of Enterococcus faecalis was 22.5% to 29.6%, while that of Enterococcusfaecium was more than 85% except for STFX (58.3%). In clinical isolates of Pseudomonas aeruginosa derived from urinary tract infections, FQs resistance was 21-27%, which was higher than that of P. aeruginosa from respiratory tract infections at 13-21%, which was the same trend as in past years. Multidrug resistant strains accounted for 5.6% in the urinary tract and 1.8% in the respiratory tract. Acinetobacter spp. showed high susceptibility to FQs. The carbapenem resistant strains, which present a problem at present, accounted for 2.7%. Neisseria gonorrhoeae showed high resistance of 86-88% to FQs. The results of the present survey indicated that although methicillin-resistant Staphylococci, Enterococci, E. coli, P. aeruginosa, and N. gonorrhoeae showed resistance tendencies, and other species maintained high susceptibility rates more than 90% against FQs, which have been used clinically for over 15 years.

The influence of the mixed inoculum on the species-identification and antimicrobial susceptibility test results was evaluated for the three automated microbial systems, WalkAway-40 (Dade MicroScan, West Sacramento, CA, USA), VITEK 2 Compact (bioMérieux, Marcy l'Etoile, France) and RAISUS (Nissui Pharmaceutical, Tokyo). In the evaluation, two different species or two different strains were mixed in serial ratios and adjusted to the inoculum cell suspension for the respective systems, and then tested for the species-identification and antimicrobial susceptibility. For the species-identification, all the three automated systems experienced incorrect identifications others from the species inoculated with higher likelihoods (> 90%), e.g. Enterobacter cloacae plus Klebsiella pneumoniae resulted in K. ornithinolytica or E. aerogenes with 93% to 97% likelihoods at the mixing ratio 9 to 1. Whereas, the mixings extended-spectrum beta-lactamase (ESBL)-producing and non-producing Escherichia coli, methicillin-resistant (MRSA) and methicillin-susceptible Staphylococcus aureus, and vancomycin-resistant (VR) and vancomycin-susceptible (VS) Enterococcus faecalis, always resulted in correct detection of a small portion of resistant cells. However, minimum ratios of resistant cells for the correct detection varied by the systems, that is, RAISUS required 70% of MRSA, VITEK 2 Compact was 8%, and the WalkAway-40 was 1%. Also, when the cell suspension of VS E. faecalis spiked with Proteus mirabilis was tested, the WalkAway-40 reported as being very rare biotype, but both VITEK 2 Compact and RAISUS reported as the test inoculum being VR E. faecalis. With these results, it can be concluded that: First, incorrect species-identifications others from the inoculated species easily occur when the inoculum contains different species even at the ratio visibly indiscernible on the primary isolation agar plate. Secondly, the automated microbial systems always intend to detect antimicrobial resistant cells in the inoculum rather than to detect major susceptible cells to prevent us from reporting very major error interpretation.

During the period from January through December 2007, a total of 1,814 stool specimens from the inpatients of nine regional hospitals in mainland of the Ryukyus, were tested to identify vancomycin-resistant enterococci (VRE). All the stool specimens were primarily cultured onto the VRE selective agar plates, and a total of 195 specimens yielded positive enterococcal growth. Of 195 isolates, VRE screening agar tests identified 106 phenotypic VRE isolates, consisting 24 isolates of Enterococcus casseliflavus, 12 of E. faecalis, 4 of E. faecium, and 66 of E. gallinarum. Then, the 106 VRE isolates were tested for vanA and vanB genes by polymerase chain reaction, the results indicating none of the isolates were positive for vanA or vanB genes. With these results, it can be concluded that, at present, mainland of the Ryukyus is VRE-free area, and it is necessary to continue careful search for incoming and spreading of VRE positive for vanA and vanB genes in Okinawa.

The newly redesigned colorimetric VITEK-2 Compact system with updated Advanced Expert System (AES) (bioMerieux, Marcy l'Etoile, France) was evaluated for its accuracy and rapidity to identify clinical isolates and to detect several antimicrobial resistances. Overall, the VITEK-2 gave 95.8% of compatibility with the reference API strips (bioMerieux) in the identifications (IDs) of Gram-positive cocci (GPC), Gram-negative rods (GNR), and yeasts. The accuracy was finally estimated to 98.3% through additional confirmatory tests. Also, >90% of IDs of GPC and GNR were obtained within 7 h of incubations. The VITEK AES correctly detected 97.7% of antimicrobial resistances, including extended-spectrum beta-lactamases, oxacillin and inducible clindamycin resistances in staphylococci, vancomycin resistance in enterococci, and penicillin and erythromycin resistances in Streptococcus pneumoniae. The most resistant isolates were identified within 12 h of incubations. In conclusion, the new colorimetric VITEK-2 Compact system with AES greatly improved its accuracy in species ID and detection of antimicrobial resistances, and it will be highly acceptable to clinical microbiology laboratory function.

We describe a case of a rare clinical disorder, platypnea-orthodeoxia syndrome. A 57-year-old man was admitted with progressive dyspnea. While breathing room air, arterial oxygen saturation decreased from 92% in a recumbent position to 83% in an upright position. Transthoracic echocardiography revealed normal left and right ventricular function, but intravenous infusion of air-blood-saline resulted in the appearance of microbubbles in the left side of the heart, indicating the presence of right-to-left shunt. Transesophageal echocardiography demonstrated a small amount of right-to-left shunting through a patent foramen ovale. Further, the left lower pulmonary vein was dilated, and contrast echocardiography revealed drainage of microbubbles mainly from the left lower pulmonary vein. A pulmonary angiogram revealed the presence of a pulmonary arteriovenous fistula. The patient underwent embolization of the arteriovenous fistula with subsequent resolution of dyspnea and hypoxemia. Platypnea-orthodeoxia syndrome is rare, and patients with this syndrome require an extensive workup to determine its cause.

Flow cytometric analysis using live/dead staining dyes was applied to discriminate yeast cells treated with autoclave and various disinfectants. One fluorogenic dye, propidium iodide cannot leak into live cells with intact membranes, whereas another fluorogenic dye, thiazole orange is permeant and can enter all cells, live and dead. Thus a combination of these two dyes can classify cell populations into live, injured and dead cells. When the yeast cells were autoclaved at 121 degrees C for 5 min, almost all the cells were classified as being dead, but some yeast strains contained a little injured cells (up to 8.5%). It was also demonstrated that none of the yeast cells autoclaved could produce a visible colony on the agar plate. The 70% ethylalcohol was the most effective disinfectant tested. Almost all the yeast cells treated were classified as being dead, and no visible colony appeared after agar plate incubation. The cell suspensions of yeast treated with 0.5% chlorhexidine contained injured cells with >95%, but no visible colony appeared. Whereas, the numbers of live yeast cells after treatment with 0.025% benzalkonium chloride were less than detectable counts (<1.0%) by flow cytometry, however, visible colonies appeared on the agar plate with 10(1) to 3.6 x 10(3) colony forming units/ml. With these results, it became apparent that the combination with fluorogenic dyes and flow cytometry can provide a reliable test method to discriminate dead and/or injured cells from the reproductive live yeast cells, and is applicable for further studies. However, it should be noted that minimum detectable levels are sometimes far different between the standard colony forming assay and the flow cytometric determination.

The TOX A/B QUIK CHEK "NISSUI" which detects both toxin A (TcdA) and toxin B (TcdB) of Clostridium difficile in stool specimens through immunochromatography was first approved to be released in Japan, and we evaluated its accuracy. In the evaluation, the TOX A/B QUIK CHEK "NISSUI" could correctly detect TcdA and TcdB in solution and in stool specimens spiked with culture broth of TcdA and/or TcdB-producing isolates of C. difficile. The minimum detectable concentrations for TcdA and TcdB were determined to be < or =0.32 ng/ml and < or =0.63 ng/ml, respectively. The TOX A/B QUIK CHEK "NISSUI" gave the consistent results with the colon-endoscopic diagnosis, that is, all the 10 stool specimens from the patients with pseudomembranous colitis were read as being positive, but negative for five patients without any C. difficile-associated disease (CDAD). Of 10 positive stool specimens, one was read as being negative by the commercially available test reagents that can detect only TcdA. In clinical evaluation, a total of 240 stool specimens were tested. Of these, the TOX A/B QUIK CHEK "NISSUI" gave 19 positive results, and TcdA and/or TcdB-producing strains of C. difficile were successfully isolated from all the positive stool specimens, except one. Whereas, of 221 negative stool specimens, 28 isolates of C. difficile were recovered and 11 isolates were identified as TcdA and/or TcdB-producing strains. With these results, it can be concluded that the TOX A/B QUIK CHEK "NISSUI" can correctly detect both TcdA and TcdB of C. difficile, and should be promptly applied to clinical microbiology laboratory to make a definite diagnosis of CDAD, particularly for the CDAD caused by the TcdA-negative but TcdB-positive mutant strains.

A total of 18,639 clinical isolates in 19 species collected from 77 centers during 2004 in Japan were tested for their susceptibility to fluoroquinolones (FQs) and other selected antibiotics. The common respiratory pathogens, Streptococcus pyogenes, Streptococcus pneumoniae, Moraxella catarrhalis and Haemophilus influenzae showed a high susceptible rate against FQs. The isolation rate of beta lactamase non-producing ampicillin-resistant H. influenzae was approximately three times as large as those of western countries. Most strains of Enterobacteriaceae were also susceptible to FQs. The resistance rate of Escherichia coli against FQs has however been rapidly increasing so far as we surveyed since 1994. The FQs-resistant rate in methicillin-resistant Staphylococcus aureus (MRSA) showed approximately 90% except for 36%. of sitafloxacin while FQs-resistant rate in methicillin-susceptible S. aureus (MSSA) was around 5%. The FQs-resistant rate of methicillin-resistant coagulase negative Staphylococci (MRCNS) was also higher than that of methicillin-susceptible coagulase negative Staphylococci (MSCNS), however, it was lower than that of MRSA. In Pseudomonas aeruginosa clinical isolates, 32-34% from UTI and 15-19% of from RTI was resistant to FQs. Acinetobacter spp. showed a high susceptibility to FQs. Although FQs-resistant Neisseria gonorrhoeae have not been increased in western countries, it is remarkably high in Japan. In this survey, isolates of approximately 85% was resistant to FQs.

The susceptibility of four macrolides (erythromycin, clarithromycin, roxithromycin and azithromycin) and a ketolide (telithromycin) was tested using 58 clinically isolated strains of the 'Streptococcus milleri' group (SMG). Among the 58 strains, 9 strains were determined to be resistant to erythromycin as well as other macrolides. Of the four macrolides and the ketolide, telithromycin was the most effective antibiotic against the SMG, including the erythromycin-resistant strains.

Recently, the instance of beta-lactamase-negative ampicillin (AMP)-resistant (BLNAR) strains of Haemophilus influenzae has exhibited a marked increase in Japan. Our group determined the MICs of 160 clinical isolates of H. influenzae at a university hospital in Okinawa, the southernmost part of Japan, and found that 27 strains were BLNAR, while 24 strains were beta-lactamase-producing. Among the latter, 2 strains were resistant to AMP/clavulanic acid. BLNAR strains were shown to be more resistant to cephems than non-BLNAR strains. The competitive affinity assay using biotinylated AMP for penicillin-binding protein (PBP) showed that the binding of cefotiam to PBP 3A/3B of BLNAR strain C2163 was lower than that of the AMP-susceptible strain, while bindings to other PBPs were not changed. The sequences of ftsI, the gene encoding transpeptidase domain of PBP 3A and/or PBP 3B, were determined, and it was found that sequences of the ftsI gene of BLNAR strains were heterogeneous mutations. Deduced amino acid sequence analyses of BLNAR strains showed that three residues (Asn-526, Val-547, and Asn-569) were replaced with Lys, Ile, and Ser, respectively. In addition, some BLNAR strains had an additional three residues (Met-377, Ser-385, and Leu-389) in ftsI replaced with Ile, Thr, and Phe, respectively. Furthermore, changes from Asp-350 to Asn-350 and from Ser-357 to Asn-357 were also found in most BLNAR strains. These substitutions were located around the penicillin binding sites of PBP3. Multiple substitutions in the amino acid sequence seemed to be closely related with extended resistance against beta-lactams, including third-generation cephems. Randomly amplified polymorphism DNA fingerprinting of clinical isolates of BLNAR strains showed genetic heterogeneity of the strains, suggesting that the prevalence of BLNAR in this region was a result of the emergence of multiple clones of this phenotype.

The in vitro activities of sitafloxacin and seven other fluoroquinolones a (ciprofloxacin, tosufloxacin, sparfloxacin, levofloxacin, T-3811ME, moxifloxacin and trovafloxacin) were examined by the microdilution method against 79 clinically isolated 'Streptococcus milleri' group (SMG) microorganisms. No statistically significant differences were found between the minimum inhibitory concentrations (MIC(50) and MIC(90)) against Streptococcus anginosus and Streptococcus constellatus. Sitafloxacin was the most active agent of the eight fluoroquinolones tested against SMG, with a MIC(90) of 0.06 microg/mL, which was 8 times more active than ciprofloxacin and 16 times more active than levofloxacin. Although none of the SMG strains showed high resistance to any of the fluoroquinolones tested, three agents (trovafloxacin, sitafloxacin and T-3811ME) had low MICs against 23 SMG strains against which levofloxacin had a MIC> 1 microg/mL. In conclusion, several fluoroquinolones have low MICs against SMG, but sitafloxacin has the lowest.

We evaluated the usefulness of an early-harvested bacterial cell suspension to the fully automated RAISUS (Nissui Pharmaceuticals Co., Ltd., Tokyo) to provide the results of species-identification and antimicrobial susceptibility testings within a day after overnight-incubation of the primary cultures. A single, well-separated colony appeared on the primary culture plate was transferred onto a blood agar or chocolate agar plates, then incubated for 3 to 6 hours. The cell suspension to the RAISUS was properly prepared to the McFarland 0.5 turbidity from the early-harvested bacterial cells. When the five ATCC reference strains, consisting of Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Streptococcus pneumoniae ATCC 49619, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853, were repeatedly tested for the species-identification, all the identification results were acceptable. Antimicrobial susceptibility tests were evaluated with the above five strains and Haemophilus influenzae ATCC 49247. The results obtained indicated that the most susceptibility test results were comparable to those MICs obtained by the standard test procedure, but some strains, in particular, H. influenzae and P. aeruginosa gave significantly discrepant MICs for certain antimicrobial agents. The significant discrepancy in MIC determinations regarded the difference of viable cell concentrations in the cell suspension prepared respectively. Through the analysis of laboratory workflow, it became to apparent that 18S to 20S of the tests were completed by 5:00 p.m., and it required to wait until 3:00 a.m. to complete 90S of the tests. With these results, the early-harvested bacterial cell suspension is applicable to species-identification by RAISUS, but it is necessary to adjust viable cell concentrations to antimicrobial susceptibility test. Also, it is urgent to reconstitute a daily workflow to improve the rapidity of RAISUS test function.

During the period from 1999 to 2003, A total of 2,255 isolates of yeast from the patients of the University Hospital of the Ryukyus were determined for their antifungal susceptibilities by the ASTY (Kyokuto Pharmaceutical Industrial Co., Ltd., Tokyo) colorimetric microdilution testing. The isolates included 1,576 strains of Candida albicans, 409 of C. glabrata, 69 of C. tropicalis, 60 of C. parapsilosis and 141 of other Candida species. A high and uniform antifungal activity of amphotericin B against all the members of Candida was demonstrated with the most minimum inhibitory concentrations (MICs) ranging between 0.25 to 2.0 microg/ml. The MICs obtained from a nearly half of the isolates included against flucytosine distributed < or = 0.125 microg/ml, but 65 (2.9%) and 43 (1.9%) isolates were interpreted as being resistant and intermediate, respectively. The activity of fluconazole markedly varied by species. The resistant ratios of C. albicans and C. parapsilosis were 0.9% and 1.7%, whereas those of C. tropicalis and C. glabrata were estimated as being 40.6% and 13.7%, respectively. The activities of miconazole and micafungin were the most potent with MICs50, 0.125 and 0.06 microg/ml. However, the MICs of C. parapsilosis against miconazole and those of C. glabrata against micafungin were significantly higher when compared to those of C. albicans. By this study, it became apparent that the activities of the presently available antifungal agents were mostly similar for the time and place, but the susceptibilities of the agents among Candida species were markedly different, particularly against azole derivatives. With this, it is necessary for clinical microbiology laboratories to determine antifungal susceptibility for the isolates of yeast and to continuously monitor the effectiveness of each antifungal agent.

The newly developed culture medium, X-SA agar medium (Nissui Pharmaceuticals Co., Ltd., Tokyo) selective for Staphylococcus aureus was evaluated for its ability to detect clinical isolates of S. aureus. Besides S. aureus, X-SA agar media allowed the growth of coagulase-negative staphylococci, Bacillus cereus and some isolates of corynebacteria. However, those species were easily distinguishable from the blue and convex colonies of S. aureus. When compared to the traditional egg yolk mannitol salt agar, selectivity for the species other than S. aureus was more specific, and growth support for S. aureus was more comparable to sheep blood agar. Also, when various phenotypic variants of S. aureus were inoculated, visible colonies of mucoid colony variants and attenuated growth variants on Mueller-Hinton agar appeared on X-SA agar plates after 24 hour-incubation, but it required 48 hour-incubation for small colony variants. The fully automated microbiology system, RAISUS (Nissui Pharmaceuticals Co., Ltd.) gave comparable species-identification and antimicrobial susceptibility test results when cell suspension directly prepared from X-SA agar media was tested. However, species-identification for phenotypic variants of S. aureus was more complicated for RAISUS testing and detection of coagulase. With these results, it could be concluded that the X-SA agar medium supplemented with chromogenic substrate is superior to the traditional selective media for the detection of S. aureus, and is widely applicable for clinical microbiology as well as food microbiology.

A fully automated microbiology system, RAISUS (Nissui Pharmaceuticals Co., Ltd., Tokyo) was evaluated to determine antimicrobial susceptibility of the isolates of Haemophilus influenzae. The test procedures were fully automated from the inoculation of the test organism through the reading of growth-endpoint on the test plates. Also, Haemophilus Test Medium (HTM) broth supplemented with oxidation-reduction color dye, Redox, assures us of enough bacterial growth and of accurate interpretation of bacterial growth. In the evaluation, the RAISUS provided 99.2% (1,012 of 1,020 testings) agreement in MIC determinations, and 96.4% (983 of 1,020 testings) agreement in category interpretations when compared to the results obtained by the NCCLS M7-A6 and M100-S14 standards. Also, ampicillin (ABPC)-resistance in H. influenzae was characterized by the RAISUS through the addition of clavulanic acid (CVA) into the test broth. Sixteen beta-lactamase-positive isolates resulted in conversion to susceptible interpretation by addition of CVA, whereas the remaining 11 isolates did not. These unaffected isolates were presumptively identified as being beta-lactamase positive, ABPC/CVA-resistant (BLPACR). Of fifty-four beta-lactamase-negative isolates, twelve were regarded as being beta-lactamase negative, ampicillin-resistant (BLNAR) due to 4 microg/ml or greater MICs against ABPC and unaffected MICs by CVA. With the results obtained, it is concluded that the RAISUS can provide comparable determinations for the isolates of H. influenzae, in both MIC determinations and category interpretations, to the NCCLS standards, and is applicable to the routine testing in clinical laboratories.

Objectives: Although transesophageal echocardiography is useful to identify various cardiovascular diseases, the semi-invasive nature of the examination hampers its routine application. We investigated whether conscious sedation using a low dose of intravenous midazoram was safe and useful during the examination., Methods: We asked 55 consecutive patients undergoing transesophageal echocardiography (44 with midazoram, mean age 59 +/- 16 years, and 11 without midazoram, mean age 62 +/- 8 years) and the examiners about the effects of midazoram using a questionnaire., Results: The mean dose of midazoram was 2.2 mg (range 1-3.5 mg). All examinations were done without any adverse events. Eighty-two percent of the patients with midazoram considered the examination under conscious sedation tolerable, whereas 91% without midazoram felt it untolerable. Most examiners felt manipulation of the echo probe was easier in patients with midazoram than in those without (69% vs 27%)., Conclusions: Conscious sedation with a low dose of intravenous midazoram facilitated and increased patient tolerance during transesophageal echocardiographic examination.

Background: Although assessment of left ventricular (LV) regional work per unit of myocardium (RWM) from the wall stress-area relationship has been proposed using M-mode echocardiography, the applicable region was limited. Anatomical M-mode is a new technique which permits the M-mode cursor to be angled in any direction on digital two-dimensional images. Our objective was to characterize regional heterogeneity of LV myocardial work using anatomical M-mode., Methods: Sixteen patients were studied: 5 with idiopathic dilated cardiomyopathy (DCM), 4 hypertrophic cardiomyopathy (HCM), and 7 controls. Digital 2-dimensional echocardiographic cineloops were acquired from the mid LV short-axis view simultaneously with high-fidelity LV pressure. Using anatomical M-mode, LV internal diameters and wall thickness (H) were determined to calculate mean wall stress (sigma) at 6 equiangular directions. The volume of region, which was given by area times H, was assumed to be constant throughout one cardiac cycle from the incompressibility of myocardium. Thus, 1/H is proportional to the regional area, and then RWM was determined as an area within the sigma-ln (1/H) loop at each direction (positive values indicated a counterclockwise loop rotation)., Results: RWM from controls were heterogeneous with the highest in the lateral segments. The 6-segment average RWM was lower in both patients with DCM and HCM than controls (3.9 +/- 1.7 and 2.1 +/- 0.3 vs. 5.5 +/- 1.4 mJ/cm(3), both p < 0.05). RWM was particularly deteriorated at septal and inferior segments in patients with DCM (2.3 +/- 0.9 and 3.0 +/- 1.5 mJ/cm(3), both p < 0.05 vs. control) and at hypertrophied anterior and anteroseptal segments in patients with HCM (0.4 +/- 0.1 and 0.8 +/- 0.6 mJ/cm(3), both p < 0.01 vs. control)., Conclusions: Anatomical M-mode enabled RWM assessment at all segments including the inferoseptum and lateral regions that had been impossible for analysis, revealing regional heterogeneity. The present method has the potential to provide additional information on myocardial mechanical condition.

A fully automated microbiology system, RAISUS newly developed (Nissui Pharmaceuticals Co., Ltd., Tokyo) was evaluated for gram-positive and gram-negative clinical isolates. In the species-identification, the RAISUS comparably identified 200 of 202 isolates (99.0%) consisting of 10 species of gram-positive cocci and of 16 species of gram-negative bacilli. When compared to the MicroScan Walk/Away, which resulted in six misidentifications, the RAISUS showed higher accuracy in species-identification. In the evaluation of antimicrobial susceptibility test, the RAISUS gave comparable minimum inhibitory concentrations (MICs) and category interpretations. Of 693 testings for gram-positive and of 721 testings for gram-negative isolates, 655 (94.5%) and 688 (95.4%) testings gave comparable interpretations to the reference microdilution test, respectively. Most identification results were reported within 5-hour incubation by RAISUS, and susceptibility test results were 4 to 7 hour-incubations. With these results obtained through laboratory-based evaluation, it became apparent that the RAISUS has enough accuracy and rapidity to determine species-identification and antimicrobial susceptibility, and is applicable to the routine testing in clinical laboratories.

A fully automated microbiology system, RAISUS recently developed (Nissui Pharmaceuticals Co., Ltd., Tokyo) was evaluated for identification of enterococci and for detection of vancomycin-resistant enterococci (VRE). When a total of 124 enterococcal isolates were tested, RAISUS correctly identified 122 (98.4%) isolates. Two isolates resulted in species-identifications disagreed with the reference but agreed as belonging to the genus of Enterococcus. When a total of fifty-seven VRE isolates confirmed to be positive for vanA and/or vanB genes were tested against vancomycin, the current RAISUS susceptibility program version 1.76 could detect 41 (71.9%) isolates of VRE as having > or = 32 microg/ml MIC for vancomycin, but one was intermediate (MIC, 8.0 microg/ml) and the remaining 15 vanB-type isolates were incorrectly interpreted as vancomycin-susceptible (MIC, < or = 4.0 microg/ml). The test program based on the algorism to determine bacterial growth in the presence of vancomycin was developed and evaluated. With this test program, all the VRE isolates positive for vanA and/or vanB genes were identified as being vancomycin-resistant or intermediate interpretation. However, eight of 19 clinical isolates of E. casseliflavus and E. gallinarum intrinsically possessing vanC gene were determined as being < or = 4.0 microg/ml MIC for vancomycin. With the influence of program revision, RAISUS became to incubate the test plate longer than with the current program, but 50% of enterococcal isolates including vancomycin-resistant and vancomycin-susceptible isolates were determined within 5 hour-incubations and 90% were within 9 to 10 hour-incubations. With these results, we can conclude that the revised test program for enterococcal isolates could rapidly and correctly identify vancomycin-resistance, and will be applicable to the routine susceptibility test in clinical laboratories.

We report a rare case of bicuspid aortic stenosis complicated by an ascending aortic aneurysm and aortic dissection of DeBakey type IIIb. A 35-year-old woman was admitted to our hospital to examine her systolic murmur identified at birth. Severe aortic stenosis, dilatation of the ascending aorta, and the narrow color flow signal in the descending aorta were detected by transthoracic echocardiography. Initially, coarctation of the descending aorta was suspected, but aortic dissection, DeBakey type IIIb, was revealed by transesophageal echocardiography. Transesophageal echocardiography is indicated when only insufficient information is available on valve and aortic morphology in patients with bicuspid aortic valve.

The positivity of beta-lactamase and antimicrobial susceptibility were determined in a total of 1,358 clinical isolates at 15 hospitals and clinics in four prefectures in southern Kyushu (Okinawa, Miyazaki, Kagoshima and Kumamoto) during the period from December 1999 to February 2000. The isolates collected comprised of 176 strains of S. aureus, 203 of H. influenzae, 102 of M. catarrhalis, 206 of E. coli, 153 of K. pneumoniae, 99 of E. cloacae, 95 of S. marcescens, 201 of P. aeruginosa, 79 of E. faecalis, and 44 of E. faecium. The frequency of CPDX resistance among E. coli in particular varied geographically, and was found to be higher in Kumamoto and Kagoshima. The strains of K. pneumoniae and E. cloacae resistant to common antimicrobial agents were particularly found in Kagoshima, and one strain of IPM-resistant E. cloacae was isolated in Miyazaki. Also, the geographical difference in the frequency of LVFX resistance among the isolates of E. cloacae was noted, the results indicating the higher prevalence in Okinawa and Kagoshima. Resistant isolates of P. aeruginosa were less common in Kagoshima, and four isolates of P. aeruginosa from Miyazaki were found to be resistant to CAZ and IPM. None of the isolates of S. aureus and Enterococcus spp. was resistant to VCM or TEIC at all. The isolates of E. faecalis resistant at high-level GM (500 micrograms/ml) and SM (1,000 micrograms/ml) were found in 27.8% and 22.8%, and those of E. faecium were 6.8% and 38.6%, respectively. Overall, the ratio of MRSA among S. aureus was 67.6%, and three isolates were resistant to ABK with no less than 8 micrograms/ml of MIC. The frequency of BLNAR (beta-lactamase-negative, ampicillin resistant) among H. influenzae isolated in Okinawa was markedly higher (isolation ratio, 37.9%) when compared with other prefectures, and the isolates of BLPACR (beta-lactamase-positive, AMPC/CVA resistant) were found only in Okinawa with a ratio of 41.6%. A total of 18 strains of ESBL defined by the NCCLS criteria (M100-S11) were isolated, eight strains of K. pneumoniae and 10 strains of E. coli. Of 18 isolates of ESBL, 13 were from Kagoshima and the remaining five were from Kumamoto.

The antimycobacterial susceptibility test method newly proposed by the Japanese Society for Tuberculosis, a proportion method on egg-based Ogawa media, was evaluated in comparison with microdilution test for Mycobacterium tuberculosis complex, BrothMIC MTB-1 (Kyokuto Pharmaceutical Inc., Tokyo). In the evaluation, five antimicrobial agents, streptomycin, ethambutol, kanamycin, isoniazid and rifampicin were included. Through repeated testings of the three reference strains against five antimicrobial agents, both test methods were found to be highly precise. All the minimum inhibitory concentrations (MICs) determined by BrothMIC MTB fell within 3 log2 dilutions, however a total of 11 MICs resulted in indeterminate(I) interpretations. Whereas, all the test results by a proportion method on Ogawa media were comparable to the expected interpretations. However, three of 48 testings resulted in undeterminable interpretations due to insufficient growth on the growth control media. A total of 127 clinical isolates of M. tuberculosis complex were tested by both methods, and 89 to 90% of the test results were comparable with each other in category interpretations. However, 7.1 to 9.4% of MICs determined by BrothMIC MTB resulted in indeterminate(I), and 0.8 to 3.1% of discrepant interpretations were observed. In conclusion, both test methods were highly precise and comparable in determining antimycobacterial susceptibility for M. tuberculosis complex. Several advantages and disadvantages in each test method were discussed.