[Isolation and identification methods of enterobacteria group and its technological advancement].

In the last half-century, isolation and identification methods of enterobacteria groups have markedly improved by technological advancement. Clinical microbiology tests have changed overtime from tube methods to commercial identification kits and automated identification. Tube methods are the original method for the identification of enterobacteria groups, that is, a basically essential method to recognize bacterial fermentation and biochemical principles. In this paper, traditional tube tests are discussed, such as the utilization of carbohydrates, indole, methyl red, and citrate and urease tests. Commercial identification kits and automated instruments by computer based analysis as current methods are also discussed, and those methods provide rapidity and accuracy. Nonculture techniques of nucleic acid typing methods using PCR analysis, and immunochemical methods using monoclonal antibodies can be further developed.

Outbreak of CTX-M-3-type extended-spectrum beta-lactamase-producing Enterobacter cloacae in a pediatric ward.

A first resistant strain of Enterobacter cloacae was isolated from a blood specimen in a pediatric patient with immature teratoma-developed sepsis after combination chemotherapy. The strain produced extended-spectrum beta-lactamase (ESBL), and the same ESBL-producing strains were detected in urine samples from other patients in the pediatric ward. All strains harbored genes for bla (CTX-M-3) by polymerase chain reaction (PCR) and sequencing analysis. Analysis of pulsed-field gel electrophoresis revealed that all strains were the same clonal type. These results suggest that ESBL-producing strains might be transmitted in the ward via contact among patients or medical staff.

Analysis of micafungin in serum by capillary zone electrophoresis.

A method is developed for measuring the concentration of micafungin, an echinocandin antifungal agent, in serum, using capillary zone electrophoresis. With a 0.1M borate buffer (pH 10.0) as the run buffer, detection is carried out at 200 nm. Pretreatment is performed by adding acetonitrile to serum (1:2) for deproteinization, allowing the supernatant fluid to be taken for measurement. The detection limit of the assay is 0.5 mg/L at a signal-to-noise ratio of 3.0. Coefficients of variation for intra- and inter-assay precision are 3.45-4.47% and 2.61-7.15%, respectively, at a nominal concentration of 5.0-25.0 mg/L. Their recovery rates are 92-110%. It is convenient for the monitoring of micafungin therapy in patients contracting clinically important deep mycoses.

[Role of a medical technologist in a nutrition support team].

The nutrition support team (NST) in our hospital not only develops clinical nutritional methods in inpatients but also performs the entire nutritional management including nutritional prescription in consultation with the physician in charge. In NST activity, clinical technologists have opportunities to report information on laboratory examination values obtained in daily work and make proposals. These opportunities are useful not only for enhancing the awareness of technologists who have focused on specific duties but also for training medical technologists. We hope that medical technologists participate in NST activity as full medical staff members in many institutions.

[A case of Mycobacterium goodii infection wifh isolation from blood and a pacemaker lead].

We report a case of blood stream infection due to Mycobacterium goodii in a patient who had an implanting pacemaker. The patient injured left thorax where the pacemaker was implanted several days before septicemia. The microorganism was isolated from both blood cultures and leads of the pacemaker. The serial isolates were identified as M. goodii by conventional biochemical methods, tobramycin susceptibility test and 16Sr-RNA sequencing. This is the first reported case of M. goodii septicemia in Japan. M. goodii is regarded as an environmental bacterium and its pathogenicity has been recognized recently. The present case suggests that its ability as a primary invader should not be underestimated, especially in a patient with indwelling devices.

Determination of meropenem by capillary electrophoresis using direct injection of serum.

Concentration determination of meropenem, a carbapenem antibiotic, using a capillary electrophoresis method by direct injection of serum samples without any pretreatment is described herein. Sodium tetraborate (25 mM)-sodium hydroxide (0.1 M) containing sodium dodecyl sulfate (90 mM) is used as a run buffer (pH 10.0). Meropenem is detected at its absorption maximum at 297 nm. Migration time of meropenem is approximately 7.2 min, and the detection limit of the assay is 2.0 mg/L at a signal-to-noise ratio of 3.0. The relative standard deviations of intra- and interassay accuracies are 3.43-8.87% and 4.28-8.54%, respectively, at a nominal concentration of 6.3-100.0 mg/L, and their recovery rates are 94-111% and 92-105%, respectively.

Further method development for measurement of linezolid in human serum by MEKC.

A method for determining linezolid concentration in human serum using micellar electrokinetic capillary chromatography by direct injection of serum is described. A borate buffer (pH 8.0) containing sodium dodecyl sulfate was used as a run buffer and detection of linezolid was performed at 250 nm (its absorption maximum). The migration time of linezolid was 5.5 min and the detection limit was 0.5 mg/l (S/N = 3). The precision and accuracy of this method was good with no interference with the detection from bilirubin, hemoglobin and chyle of high concentrations. This provides a simple and easy method where samples of micro-quantity are used.

Development and validation of a MEKC method for the direct determination of cefozopran in human serum.

A method for determining the concentration of cefozopran, a cephem anti-microbial agent which has a broad spectrum, in human serum using micellar electrokinetic capillary chromatography (MEKC) by serum direct injection is developed and the validation of the assays of this method is performed. A borate buffer (25mM; pH 10.0) containing sodium dodecyl sulfate (SDS) (50mM) is used as a run buffer. The electrophoresis of serum samples is carried out at 25kV and the detection of cefozopran at 244nm as its absorption maximum at the cathodic side of the capillary. The migration time of cefozopran is 6.5min. Linearity (0-200mg/l) is good and limit of quantification is 0.5mg/l at a signal-to-noise ratio of 3. Coefficient of variation (CV) of intra-day precision and that of inter-day precision are 2.4-4.0% (7.3-92.0mg/l) and 2.9-7.7% (22.5-71.4mg/l), respectively, and the recovery rate is 92-109%. The detection results of 12 other cephem anti-microbial agents under the analytical conditions of this method show that the migration time of cefmetazole is identical with that of cefozopran, making it impossible to separate these two anti-microbial agents. This method is characterized by the fact that simple and economic determination can be achieved by directly injecting the serum samples of micro-quantities into the capillary.

Method development for determining the iohexol in human serum by micellar electrokinetic capillary chromatography.

Iohexol is widely used in clinical laboratories as a non-ionic radiographic contrast medium. Determination of its concentration in blood has a vital meaning in preventing its side effects caused by its retention in the system. A method for determining iohexol in serum by micellar electrokinetic capillary chromatography (MECC) requiring no pretreatment is developed. Electrophoresis is performed for serum samples at 25 kV with a borate buffer (50mM; pH 9.5) containing sodium dodecyl sulfate (50mM) and detection is carried out at 245 nm. Migration time of iohexol is 7.4 min. Linearity (0-1000 mg/l) is good and detection limit is 0.5mg/l (S/N=3). CV of intra-assay precision at a measurement concentration range of 6.2-200.1mg/l is 1.38-4.68% and recovery rate is 96-102%. CV of inter-assay precision is 2.06-5.94% at a measurement concentration range of 10.3-155.4 mg/l. This method is characterized by determination through direct injection of serum samples of super micro-quantity into the capillary, which simplifies the determination procedure in a significant manner and improves the precision and accuracy of determination.

A new type of temperature-dependent serum M protein: a case of IgG-lambda type multiple myeloma.

BACKGROUND: We report a rare case of temperature-dependent serum M protein (thermoprotein), monoclonal IgG(1)-lambda protein isolated from 90-year-old female with advanced multiple myeloma. METHODS: M protein was identified in the blood plasma of the patient by immunoelectrophoresis (IEP). To evaluate the types of bonds, the properties of the protein after reduction and chemical treatment were examined. RESULTS: This protein was irreversibly precipitated at or above room temperature when exposed in the air. This protein was redissolved by 30 mmol/l dithiothreitol, 4 mol/l urea, or 8 mmol/l EDTA. CONCLUSIONS: Unlike other immunoglobulins reported to date, this data suggests that hydrogen, disulfide, and ionic bonds are involved in the temperature-dependent precipitation of this M protein.

A new fungal lectin recognizing alpha(1-6)-linked fucose in the N-glycan.

In this report, we describe a new lectin from the fungus Rhizopus stolonifer that agglutinates rabbit red blood cells. Agglutinating activity was detected in the extract of mycelium-forming spores cultured on agar plates but not in the mycelium-forming no spores from liquid medium. This lectin, which we designated R. stolonifer lectin (RSL), was isolated by affinity chromatography with porcine stomach mucin-Sepharose. SDS-polyacrylamide gel electrophoresis and mass spectral analysis showed that RSL is approximately 4.5 kDa, whereas gel filtration indicated a mass of 28 kDa. This indicates that the lectin is a hexamer of noncovalently associated RSL monomers. RSL activity was very stable, since it was insensitive to heat treatment at 70 degrees C for 10 min. Analysis of RSL binding specificity by both microtiter plate and precipitation assays showed that N-glycans with l-fucose linked to the reducing terminal GlcNAc residues are the most potent inhibitors of RSL binding, whereas N-glycans without alpha(1-6)-linked fucose residues are approximately 100-fold weaker inhibitors of binding. Oligosaccharides with alpha(1-2, -3, and -4) linkages showed no inhibition of binding in these assays. In a mirror resonance biosensor assay, high affinity binding was observed between RSL and the glycopeptide of bovine gamma-globulin, which has N-glycans with alpha(1-6)-linked fucose residues. However, RSL showed only a weak interaction with the glycopeptide of quail ovomucoid, which lacks fucose residues. Finally, capillary affinity electrophoresis studies indicated that RSL binds strongly to N-glycans with alpha(1-6)-linked fucose residues. Together, these results show that RSL recognizes the core structure of N-glycans with alpha(1-6)-linked l-fucose residues. This specificity could make RSL a valuable tool for glycobiological studies.

Determination of antibacterial flomoxef in serum by capillary electrophoresis.

A determination method of flomoxef (FMOX) concentration in serum by capillary electrophoresis is developed. Serum samples are extracted with acetonitrile. After pretreatment, they are separated in a fused-silica capillary tube with a 25 mM borate buffer (pH 10.0) as a running buffer that contains 50mM sodium dodecyl sulfate. The FMOX and acetaminophen (internal standard) are detected by UV absorbance at 200 nm. Linearity (0-200 mg/L) is good, and the minimum limit of detection is 1.0 mg/L (S/N = 3). The relative standard deviations of intra- and interassay variability are 1.60-4.78% and 2.10-3.31%, respectively, and the recovery rate is 84-98%. This method can be used for determination of FMOX concentration in serum.

[Norwalk virus and Noro virus].

Norwalk virus and Noro virus are members of the Caliciviridae. These viruses are morphological similarity in each other and shows small round structure. These viruses also are well known as main pathogens of acute infectious gastroenteritis. Clinical features include an incubation period of 24 of 48 hours and illness period of 18 to 72 hours with vomiting and diarrhea in most patients and high secondary attack rates. Oral transmitted infection occurs contaminated water and foods. In our country, outbreak of Noro virus-related gastroenteritis are reported sometimes in hospital and nursing home from winter to early spring seasons. This article are described to the morphlogy, physical characteristics, epidemiology, and clinical manifestation relating to Norwalk virus and Noro virus.

Method development for determining the antibacterial linezolid in human serum by micellar electrokinetic capillary chromatography.

A precise method for determining linezolid concentration in human serum by micellar electrokinetic capillary chromatography has been developed and validated. Serum was deproteinized with acetonitrile and etofylline was used as an internal standard. A borate buffer (pH 10.0; 25 mM) containing sodium dodecyl sulfate (80 mM) was used as a running buffer. Detection was performed at UV253 nm by applying 25 kV voltage to a fused-silica capillary tube. Migration time of linezolid was approximately 14 min. Good linearity (0-100 mg/l) was obtained and the limit of detection was 0.5 mg/l (S/N=3). This technique covered the clinical concentration (4 mg/l) measurement of this drug enough. The intra- and inter-day reproducibility was good. Serum recovery was 95-102%. No interference from other anti-microbial agents was observed. Linezolid after serum deproteinization showed high stability. This method was easy to operate as well as economical as a method for determining linezolid in serum.

[Mycoses].

Antifungal susceptibility testing has developed rapidly during the last decade. Through the intensive collaborative work, the National Committee for Clinical Laboratory Standards (NCCLS) has published a standardized antifungal susceptibility method M27-A which included interpretive guidelines for 3 antifungal agents. Meaningful large-scale, longitudinal studies of antifungal susceptibility and resistance, based on standardized methodology and interpretive guidelines, have been published and serve as the basis for analysis of resistance trends. Resistant isolates have been further characterized using molecular biological techniques. Candida albicans and related species become resistant to antifungal agents, in particular triazoles, by expression of efflux pomps that reduce drug accumulation, alteration of the structure or concentration of antifungal target enzymes, and alteration of membrane sterol composition. On the other hand, there are several ongoing problems in antifungal susceptibility testing such as trailing phenomenon, ability to detect amphotericin B resistance, and application for other fungal pathogens. In this paper, we review studies which focused on the reliability of antifungal susceptibility testing, epidemiology of resistant isolates, and resistant mechanisms. Furthermore, we provide several recommendations for antifungal susceptibility testing in the clinical laboratory.

Quantification of pilsicainide in serum by capillary electrophoresis.

A new method for determining pilsicainide concentration in serum by rapid and selective capillary electrophoresis has been developed and validated. For pretreatment, serum was made alkaline and then extracted with diethyl ether. Procainamide was used as an internal standard. Sodium dihydrogenphosphate buffer (pH 2.29; 0.1 M) was used as a running buffer. A fused-silica capillary tube was loaded with a voltage of 25 kV and detection was performed at UV 200 nm. Good linearity (0-2.5 microg/ml) was obtained with the minimum limit of detection being 0.04 microg/ml serum (signal-to-noise ratio, 3:1). The R.S.D. of within-run reproducibility was 0.798-2.32%, that of between-run reproducibility was 4.74-5.12% and the recovery rate was 61-63%. Disopyramide, another anti-arrhythmic drug, was close to pilsicainide in terms of migration time. This method was applied to determination of pilsicainide in serum samples.

[Diagnosed tuberculosis using specific DNA probe hybridization methods].

In Japan, reported cases of tuberculosis had declined nearly every year until 1995. However, in 1997 newly recorded cases began increasing for the first time in more than 38 years. Recent studies using DNA fingerprinting show that person- to person transmission may account for as many as one-third of new cases of tuberculosis in citizen populations. Nucleic acid hybridization methods using specific DNA probes can specifically identify M. tuberculosis and other mycobacterial species. Rapid nucleic acid amplification techniques such as polymerase chain reaction methods allow direct identification of M. tuberculosis in clinical specimens. Is 6110 has been exploited extensively as a clonal marker in molecular epidemiology studies of tuberculosis. The emergence of resistance to antituberculosis drugs is a relevant matter worldwide. A recent genotypic method allows earlier detection of RFP-resistant and INH-resistant stains using probes for mutation in rpoB and in katG.