Current practice of management of bacteremic sepsis: a study in a tertiary care teaching hospital in Japan.

OBJECTIVE: To investigate how patients with bacteremic sepsis are managed in a tertiary care teaching hospital. PATIENTS AND METHODS: Prospective observational study on patients with bacteremic sepsis. Clinical and microbiological characteristics of bacteremic sepsis were analyzed in relation to prognosis. Severity of the illness was quantitatively analyzed by the APACHE (Acute Physiology, Age, Chronic Health Evaluation) III scoring system. Also investigated was how closely physicians paid attention to acute physiological alterations in patients. RESULTS: The 28-day mortalities in fifty hemodynamically stable patients and in twenty-three septic shock patients were 26% and 52%, respectively (p=0.028). Gram-positive organisms accounted for 54% of all organisms, with the mortality and incidence of septic shock being the same as with Gram-negative infections. The mean APACHE III score was 42.9 in survivors, and 76.5 in non-survivors (p < 0.001). Although serum levels of C-reactive protein and acute physiology score (APS) was significantly higher in non-survivors than in survivors, the correlation with APACHE III score was more prominent in APS. The number of vital signs recorded was 1.67 in physicians and 3.6 in nurses (p < 0.001). CONCLUSIONS: The present study proved that the APACHE III score accurately discriminates between survivors and non-survivors of patients with sepsis. By addressing the need for an objective evaluation of severity of illness, it strongly recommends that physicians should be made aware of physiologically defined sepsis and that they should pay closer attention to patients' physiological alterations to identify the development of sepsis in critically ill patients.

[A trial of infection control measure by clinical microbiology laboratory].

In 1998, we developed a Total Infection Control System in Saga Medical School Hospital, and would like to introduce it for the practical use. This system was named "Dr. FLEMING" (Flexible Microbiological Test & Information System for the New Generation) and is expected to help physicians by providing highly valuable test results and useful information. For example, bacterial identification and drug susceptibility test can be completed within 4-6 hrs after bacterial colony is isolated, and the test report the contains full-colored pictures to enhance understanding. In addition, we have made an information center for infectious disease, where physicians can have access to various data bases outside our hospital. Furthermore, we offer many kinds of useful information to physicians working at other medical facilities to assist their clinical practice of infectious diseases.

Mutational analyses of restriction endonuclease-HindIII mutant E86K with higher activity and altered specificity.

We have performed mutational analyses of restriction endonuclease HindIII in order to identify the amino acid residues responsible for enzyme activity. Four of the seven HindIII mutants, which had His-tag sequences at the N-termini, were expressed in Escherichia coli, and purified to homogeneity. The His-tag sequence did not affect enzyme activity, whereas it hindered binding of the DNA probe in gel retardation assays. A mutant E86K in which Lys was substituted for Glu at residue 86 exhibited high endonuclease activity. Gel retardation assays showed high affinity of this mutant to the DNA probe. Surprisingly, in the presence of a transition metal, Mo(2+) or Mn(2+), the E86K mutant cleaved substrate DNA at a site other than HindIII. Substitution of Glu for Val at residue 106 (V106E), and Asn for Lys at residue 125 (K125N) resulted in a decrease in both endonucleolytic and DNA binding activities of the enzyme. Furthermore, substitution of Leu for Asp at residue 108 (D108L) abolished both HindIII endonuclease and DNA binding activities. CD spectra of the wild type and the two mutants, E86K and D108L, were similar to each other, suggesting that there was little change in conformation as a result of the mutations. These results account for the notion that Asp108 could be directly involved in HindIII catalytic function, and that the substitution at residue 86 may bring about new interactions between DNA and cations.

[The new generation of laboratory automation systems].

Laboratory automation is essential to release laboratory technicians from simple routine work, allowing them to make use their time for more skilled tasks. In 1998, we developed a Total Infection Control System called "Dr. Fleming", and would like to introduce it into practical use. This systems is expected to help physicians by providing highly valuable test results and useful information.

Protein-bound polysaccharide PSK inhibits tumor invasiveness by down-regulation of TGF-beta1 and MMPs.

Transforming growth factor beta1 (TGF-beta1) and matrix metalloproteinases (MMPs) produced by tumor cells play important roles in tumor invasion. PSK, a protein-bound polysaccharide, is widely used in Japan as an immunopotentiating biological response modifier for cancer patients. In this study, we focused on the effects of PSK on invasiveness, TGF-beta1 production, and MMPs expression in two human tumor cell lines, pancreatic cancer cell line (NOR-P1) and gastric cancer cell line (MK-1P3). PSK significantly decreased the invasiveness of both cell lines through Matrigel-coated filters but did not affect cell viability, proliferation, or adhesion. Decreased invasion was associated with the inhibition of TGF-beta1, MMP-2, and MMP-9 at both mRNA and protein levels as assessed by reverse transcriptase-polymerase chain reaction, gelatin zymography, and enzyme-linked immunosorbent assay. Antibody against TGF-beta1 neutralized the MMP activities of both cell lines. PSK also suppressed the expression of urokinase plasminogen activator (uPA) and uPA receptor but did not change plasminogen activator inhibitor-1 (PAI-1) expression. Western blot analysis showed that PSK reduced uPA protein expression but not PAI-1 expression in the both cell lines. These results indicate that PSK suppresses tumor cell invasiveness through down-regulation of several invasion-related factors including TGF-beta1, uPA, MMP-2, and MMP-9.

Site-directed mutagenesis of restriction endonuclease HindIII.

Site-directed mutagenesis by inverse PCR was done on the HindIII gene. Target residues to be mutated were chosen according to (i) the fact that a mutant obtained by sodium nitrite treatment showed almost no HindIII activity, where Asp-123 was replaced with Asn, and (ii) the model proposed by Stahl et al. (Stahl, F., Wende, W., Jeltsch, A. and Pingoud, A. Biol. Chem. 379, 467-473 (1998)). Seven kinds of mutants were obtained by the PCR, and their enzymatic and biochemical properties were examined. Three mutants, P50S, D108L, and D123N, showed fairly low HindIII activity. On the other hand, the other four, P84Q, E86K, V106E, and K125N, retained the activity. In particular, E86K showed higher activity than the wild enzyme. This fact was confirmed when activities of the purified wild and E86K enzymes were assayed. These results coincided fairly well with data using E. coli strains that carry the respective mutant plasmids, on their resistance to phage T7 and on growth rate. We conclude that the PE motif at residues 50 and 51, and DXK motif at residues 108-110, are responsible for the enzymic reaction of HindIII.

Prevalence and genetic diversity of cagD, cagE, and vacA in Helicobacter pylori strains isolated from Japanese patients.

BACKGROUND: Although cagD and cagE (cagDE) identified upstream of cagA have been shown to be involved in the induction of interleukin (IL)-8 expression, the relationship between cagDE status and gastroduodenal diseases still remains to be examined. Thus we investigated prevalence and genetic diversity of cagD, cagE, and vacA in Helicobacter pylori strains isolated from patients with peptic ulcer or gastritis. METHODS: We analyzed 73 H. pylori strains isolated from Japanese patients (gastritis (GA), 15; gastric ulcer (GU), 28; duodenal ulcer (DU), 23; GU and DU, 7). The presence of cagDE was evaluated by polymerase chain reaction (PCR) and Southern hybridization. The vacA genotype was examined by PCR, using type-specific primers. RESULTS: cagDE was present in 13 (86.7%) of 15 patients with GA, 26 (92.9%) of 28 patients with GU, 21 (91.3%) of 23 patients with DU, and 6 (85.7%) of 7 patients with GU and DU (P = 0.89). vacA signal sequence type s1 was found in 14 (93.3%) of 15 patients with GA, 26 (92.9%) of 28 patients with GU, 22 (95.7%) of 23 patients with DU, and 6 (85.7%) of 7 patients with GU and DU (P = 0.84). Sequences of cagDE and vacA in our Japanese strains were highly homologous with one another, and there were no disease-specific mutations. CONCLUSIONS: Most of the H. pylori strains in Japan were cagDE-positive, vacA s1 type, regardless of clinical outcome. The present study also indicated that these genes were conserved well among our H. pylori isolates.

[The introduction of automation and systematization in the department of microbiology].

The Department of Laboratory Medicine is considered to be the transmission subdivision of medical information. However, most of the information is provided from the subdivisions of clinical chemistry, hematology and immunology. Moreover, the information is slowly transferred from the subdivision of microbiology. The Department of Laboratory Medicine at Saga Medical School developed a flexible microbiological test & information system in 1998, called Dr. Fleming. However, many methods of operation, effects, and problems are associated with the standardization and introduction of such a system. Therefore, the ideas of leaders in the field of microbiology and infectious disease will be discussed before establishing this system.

Education as a means of reducing medical expenses.

In treating patients with infectious diseases, key knowledge and experience are essential in making appropriate clinical decisions. Medical students in Japan receive limited tuition in clinical pharmacology and microbiology in their undergraduate curriculum. Education and guidance in these fields are not always provided, even during postgraduate training. To help overcome this problem, we have devised a quick medical reference system to support antimicrobial chemotherapy, and this has been operating in our hospital since May 1994. This system is integrated in order to convey the maximal significance of test results by providing detailed information on various kinds of pathogens and antibiotics immediately on every computer display. This is a unique system in Japan, and aims to help doctors provide effective therapy. Using this system, we have succeeded in reducing medical expenditure for antimicrobials by around 10%.

Necrotizing fasciitis caused by Vibrio vulnificus differs from that caused by streptococcal infection.

We reviewed the clinical record of all patients admitted to Saga Medical School Hospital during the most recent 10 years and found that 17 (0.03%) were diagnosed as having necrotizing fasciitis. Bacteriological examination demonstrated that Vibrio vulnificus was the pathogen responsible in five patients (29%). The disease caused by V. vulnificus occurred in the warmer half of the year. All of the patients had underlying chronic liver dysfunction, and three of them had previously consumed raw seafood. In these patients, the predominant skin lesions were oedema and subcutaneous bleeding, such as ecchymosis and purpura, while superficial necrosis was not recognized. Three patients died of systemic complications. By contrast, all of the five patients with necrotizing fasciitis caused by Streptococcus pyogenes had the disorder in winter, and only one of them had chronic liver dysfunction. In skin lesions, subcutaneous bleeding was rare but necrosis was seen often. Despite the high incidence of systemic complications, no patients with streptococcal necrotizing fasciitis died. These findings suggest that the clinical features of necrotizing fasciitis caused by V. vulnificus are different from those of necrotizing fasciitis caused by classical pathogens, and that the two should be differentiated as early as possible to improve the prognosis.

Refractory anaemia with ringed sideroblasts concurrent with multiple myeloma--a brief review of the recent literature.

The report describes a patient in whom myelodysplastic syndrome and multiple myeloma (MM) were simultaneously present. This patient manifested an IgA-lambda type of MM concurrent with a refractory anaemia with ringed sideroblasts (RARS) without prior therapy. His bicytopenia could not be improved by vitamin B6 regardless of a reduced serum vitamin B6 concentration. A review of the literature suggests that myelodysplastic syndrome (MDS), chronic neutrophilic leukaemia (CNL) and idiopathic myelofibrosis (IMF) are the most frequent disorders associated with MM. The IgA type seems to be associated more commonly with these disorders. The mechanisms responsible for the development of plasma cell proliferation are diverse; the neoplastic transformation of a common progenitor, the involvement of the lymphoplasmacytic system and/or chronic reticuloendothelial stimulation may play a role in the occurrence of such hybrid haematological disorders.

Anionic properties of beta-lactam-enhancing factor on methicillin-resistant Staphylococcus aureus.

We found a Factor (factor T) in aged mixtures of tungstate and phosphate which greatly enhanced the antibacterial effects of beta-lactams upon methicillin-resistant Staphylococcus aureus (MRSA). Factor T suppressed penicillinase production and the amount of penicillin-binding protein-2' in the membrane fraction, thus sensitizing MRSA strains to beta-lactams. In addition, Factor T caused a metachromatic reaction and prolonged the blood coagulation time, indicating that it is a heparin-like polyanion. Since Factor T becomes ineffective in the presence of a polycation, a charge interaction may play an important role in the enhancing effect. One possibility is that Factor T non-specifically inhibits several anion-sensitive enzymes. Factor T inhibited several nucleotide-interacting enzymes, but not most serum enzymes.

[The Quick Return Service of electroencephalogram].

Because electroencephalography (EEG) examinations take time and numerous channels are used, they generate an enormous volume of data. Vast amounts of space would be required to store the data on shelves and in storage rooms, and a great deal of time and labor would be needed to retrieve the data and use it again. We developed an EEG optical disk filing system to solve such problems. With this system display terminals are set up in the various outpatient departments which often request EEG examinations, i.e., the neurology department, psychiatry department, and pediatric department, making it possible to search and display EEG waveforms, the content of EEG reports, etc., stored on the optical disks at any time. Using such functions patients can be examined without having to send for charts and EEG paper-output waveforms, which is troublesome and time-consuming, and contributes to shortening examination time.

Characterization of humoral and cellular immunity in the central nervous system of HAM/TSP.

In HTLV-I-associated myelopathy or tropical spastic paraparesis (HAM/TSP) immunopathological processes in the central nervous system (CNS) have not been clarified. We compared the humoral and cellular immunity within the CNS and in the systemic circulation of 24 patients with HAM/TSP (8 men and 16 women) to 6 asymptomatic HTLV-I carriers, 7 patients with active multiple sclerosis, 6 patients with acute viral encephalitis, and 39 patients with other non-inflammatory neurological diseases. Significant differences were observed between the HAM/TSP patients and one or more of the control groups: HAM/TSP cerebrospinal fluids (CSF) exhibited higher levels of IgG, IgG index, de novo IgG synthesis rate, and beta 2-microglobulin, and also a predominance of CD8+ cells that expressed CD11a and CD45RO but lacked CD28 antigens. Results in the 6 patients with acute viral encephalitis suggested that the CD8+ population in the CSF which is positive for CD28 and CD45RO is important for the elimination of virus from infected CNS tissues. Therefore, potentially cytotoxic T cells of a unique CD8+CD11a+CD45RO+CD28- phenotype may play a key role in the CNS pathogenesis of HAM/TSP.

[Peripheral T-cell lymphoma with abundant ATL-like cells in the blood].

A 69-year-old woman was admitted to our hospital because of leucocytosis and systemic lymphadenopathy. On admission, white blood cell count was 163,000/microliters, most of which consisted of flower-like cells with convoluted nuclei in the peripheral blood. In the abnormal lymphocyte cells surface-marker test by flow cytometry showed that they expressed CD2, CD3, CD4, CD29, CD45RA, and CD38, but not CD8, CD16, and CD25. Serum anti-Human T-lymphotropic virus type-I (HTLV-I) antibody was negative in particle agglutination, enzyme-linked immunosorbent assay (ELISA) and western-blotting assay. HTLV-I proviral DNA in the abnormal lymphocyte cells was not detected by southern blotting hybridization technique. Moreover, HTLV-I provirus was not detected using a polymerase-chain-reaction (PCR). A monoclonal rearrangement of the TCR-beta chain gene was evident by using DNA probe in southern blot hybridization. Because of the rapid progress of the disease, chemotherapy was started immediately after admission. Though, this patient became refractory, and she died about 1 year after admission.

Intracellular multiplication of Legionella pneumophila in HL-60 cells functionally differentiated in response to 22-oxacalcitriol.

The agent 1,25-dihydroxyvitamin D3 (D3) induces the differentiation of HL-60 human leukemia cells into functional monocyte-like cells that can support the intracellular multiplication of Legionella pneumophila. 22-Oxacalcitriol (OCT), a synthetic analogue of D3, exhibits greater differentiation-inducing activity than D3 in WEHI-3 mouse leukemia cells and has been suggested to be clinically more useful because of its lower hypercalcemic activity. The abilities of OCT and D3 to induce the functional differentiation of human leukemia HL-60 cells have now been investigated. OCT induced the differentiation of HL-60 cells into monocyte-like cells to a similar extent as D3. Thus, both OCT and D3 increased (1) the surface expression of CD11b, CD11c, CD14, and CD35; (2) nonspecific esterase staining; and (3) phagocytic activity toward fluorescent beads. HL-60 cells differentiated in response to OCT also supported the intracellular multiplication of L. pneumophila. Activation of both OCT- and D3-treated HL-60 cells with human recombinant interferon-gamma (IFN-gamma) for 24 h before infection markedly inhibited L. pneumophila multiplication. IFN-gamma activation enhanced superoxide anion generation by D3-treated HL-60 cells but not by OCT-treated HL-60 cells, suggesting that the inhibition of L. pneumophila multiplication in IFN-gamma-activated cells is independent of superoxide generation. Finally, D3, but not OCT, markedly stimulated the formation of osteoclast-like multinucleated cells from mouse bone marrow cells, consistent with the lower hypercalcemic activity of OCT.