Genomic DNA from 56 Vibrio cholerae 0139 strains isolated in various countries was digested with Sfi I or Not I and analyzed by pulsed-field gel electrophoresis (PFGE). Eight different PFGE patterns were identified. Although the patterns of a large majority of CT-genepositive epidemic strains isolated in India, Bangladesh and Thailand were the same or similar, but were slightly different from those of two CT-positive strains from India and Nepal. On the other hand, the patterns of CT-negative three strains from Argentine, SriLanka and Bangladesh were apparently different not only from each other, but also from those of epidemic CT-positive strains. The pattern of one V. cholerae 01 El Tor strain isolated in India and the pattern of epidemic V. cholerae 0139 resemble each other in many points.

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Recently, nosocomial outbreaks of MRSA have become an important social problem in Japan. To examine the routes of transmission of MRSA, the establishment of accurate MRSA typing system is essential. However, more recently, because MRSA strains with type II coagulase have been increasing, it is difficult to discriminate MRSA strains by the coagulase typing method. Under this background, our study was designed to evaluate the clinical significance of DNA fingerprinting by arbitrarily primed polymerase chain reaction (AP-PCR). Several MRSA strains isolated from patients in our department were used in this study. To optimize the condition of AP-PCR, the differences of amplified products by AP-PCR were evaluated according to the following conditions: extracting methods of DNA from MRSA strains, buffer conditions, the temperature of AP-PCR, cycles of AP-PCR, and several primers. As a result, the optimal conditions of AP-PCR were as follows: extracted DNA using the Insta Gene kit, amplified DNA by two-step AP-PCR using a M13 reverse primer with a buffer condition of 3.5 mM of magnesium chloride, and a pH of 8.5. The results of AP-PCR correlated well with the results of pulsed-field gel electrophoresis. In conclusion, DNA fingerprinting by AP-PCR seems to be useful in examining the nosocomial MRSA outbreak.

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For 10 strains of Neisseria gonorrhoeae exhibiting decreased susceptibility to new quinolones (MIC≥0.1μg/ml) isolated and preserved from patients with gonococcal urethritis during the period from February 1991 through January 1992. We investigated the mechanism for development of resistance to new quinolones. After selecting 3 PCG-sensitive and new quinoloneresistant strains from among these strains, we first carried out transformation experiments of N. gonorrhoeae using a plasmid carrying the E. coli gyrA gene. We then determined the base sequence on the N. gonorrhoeae gyrA gene by PCR method. Of the 3 strains in which tranformation experiments were carried out, 2 strains yielded transformants, one of which was 8 times more sensitive to norfloxacin (NFLX) than the original strain, and it was assumed that this strain has a mutation in the gyrA gene. In our study of the base sequence on the N. gonorrhoeae gyrA gene using the PCR method, both strains had the mutation of Ser 83 (TCC)→Phe (TTC), and in addition to this mutation of Ser→Phe, one other strain had the mutation of Asp-87 (GAC)→Gly (GGC). This type of mutation of quinolone-resistant N. gonorrhoeae on the gyrA gene has high homology with the mutations reported for Escherichia coli and other strains and there appears to be a close correlation between the increasing frequency of use of the new quinolones in this country and the spread of such resistant strains of N. gonorrhoeae.

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We have performed serial bronchoalveolar lavage (BAL) examination in two patients with asperigilloma and compared that of total cell count and cell population with control groups (5 non-smokers, 10 smokers) and other pulmonary infectious diseases: 7 each with mycoplasmal pneumonia and pulmonary tuberculosis, 6 with bacterial pneumonia, and 5 with chlamydial pneumonia.
Miconazole (MCZ) by drip intravenous infusion of 400 mg/day and flucytosine (5-FC) by oral intake of 4.5 to 6.0 g/day were administered to one patient with aspergilloma, and we studied the serum and BALF concentration about 5 hours after administration.
The followings results were obtained:
1. In aspergilloma, the cell population of neutrophils in BALF increased compared with control groups (p <0.01) and other pulmonary infectious disease.
2. The serum and BALF levels of MCZ ranged from 0.1 to 0.3μg/ml, <0.1 to 14.4μg/ml, respectively. On the other hand, the serum and BALF levels of 5-FC ranged from <0.2 to 9.3 item', and <0.4 to 1.5μg/ml, respectively.
From these results, we consider that neutrophils play the main role in the immune host defense in aspergilloma, and the combination of intracavitary infusion of MCZ and oral administration of 5-FC should be the treatment of choice.

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We carried out a survey for the prevalence of antibodies to the spotted fever group (SFG) rickettsia in vertebrates such as dogs, cattle, deer, and mice in Shimane Prefecture. Rickettsia japonica was employed as antigen in indirect immunofluorescence (IF) tests. The experiment for natural infections using decoy animals was performed in the field of the endemic area.
1. Among 115 street dogs, 18.3% possessed the antibodies against SFG rickettsia, while all of the 8 hunting dogs had the antibodies.
2. Among 234 cattle tested, 17.9% possessed the antibodies. IF titers were 1: 40 to 1: 160 (mean 1: 68).
3. Among 69 wild deer, 92.7% possessed the antibodies ranging between 1: 40 and 1: 640, which showed the highest IF titers (mean 1: 89) among those of the examined vertebrates.
4. The incidence of the antibodies in Apodemus speciosus, Apodemus argenteus and Eothenomys smithi smithi mice were 16.5, 4.3 and 0%, respectively. The incidence of the antibodies against SFG rickettsia in mice captured in the endemic area was significantly higher (22.8%) than that in non-endemicarea (10.4%). Difference in the incidence of antibody-positive mice was also observed within the endemic area. Therefore, we concluded that the infection of mice was restricted to a limited area.
5. No significant rise in IF titers was observed in decoy animals that had been infested with ticks in the endemic area.

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We devised a micro-suspension-test to evaluate disinfectants against human immunodeficiency virus type 1 (HIV-1) and confirmed its reliability. Suspensions of persistently HIV-1-infected Molt-4 cells were used as targets of disinfectants and residual infectivity was measured by an infectivity assay: after cocultivation with uninfected Molt-4 cells reverse transcriptase activity (RTA) in the supernatant and giant cell formation (GCF) were monitored. Our new infectivity assay consists of a short-term assay, that is RTA and GCF monitoring on the second day of co-culture, and a long-term assay, that is RTA monitoring up to the 28th day of co-culure. The sensitivity of the short-term assay was 1×10³ infected cells and that of the long-term assay was 1×10¹ infected cells. All the chemical disinfectants examined in this study showed dose-and time-dependent inactivation of HIV-1. By 5-minute contact with ethanol, glutaraldehyde, formalin, sodium hypochlorite and povidone-iodine, HIV-1 was effectively inactivated at concentrations of 20, 0.01, 5, 0.05 and 0.1%, respectively. Since the micro-suspension-test is easy and sensitive, we recommend it as a method for evaluating disinfectants against HIV-1.

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Accuracy amplified Mycobacterium Tuberculosis Direct Test (MTD), Amplicor Mycobacteria kit (Amplicor) and PCR method routinely used in the University of Tokyo Hospital (J. Clin. Microbiol. 31: 446-450 1991) were evaluated and compared with the same samples.
The detection limits of MTD, Amplicor and PCR method (University of Tokyo) were 0.01-0.1 CFU/tube, 0.625 CFU/tube and 0.2 CFU/tube respectively, which were almost the same. These were shown to be at least as sensitive as the conventional culture techniques. The Tokyo Univ. method using radioisotope, takes up to 3 days, on the other hand 2 kits take 4-5 hours. These kits become useful tools for the early and rapid detection of M. tuberculosis in uncultured clinical specimens.
But the risk of laboratory contamination and false-positive results remain. These must be further improved.

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A new recombinant parvovirus B19 antigen was tested whether it was responsive to human serum antibodies in every epidemic year of erythema infectiosum for 25 years, because wild strains of B19 parvovirus were changeable genetically. The antigen was empty particles of both B19-VP1 and VP2 produced in baculovirus expression system. Specimens were 21 sera in 1968, 19 in 1980, 44 in 1987 and 33 in 1992, derived from 67 patients with erythema infectiosum, fever and/or non-specific exanthem and aplastic crisis in persons with hereditary spherocytosis. Each patient had been confirmed of B19 parvovirus infection by other methods as radio immunoassay and/or enzyme-linked immunosolvent assay for B19-IgG and IgM using other antigens and by detection of B19-genome DNA using the polymerase chain reaction. Days of the illness of every serum were confirmed including before infection to 216 days after onset. Sera from 23 patients with measles, Kawasaki disease and rubella were selected for controls, and those patients who had not been infected with B19 parvovirus. Tests were carried out by enzyme immunoassay, indirect method for IgG and IgM capture method. In a total of 103 specimens after onset of symptoms B19-IgG was positive in yearly specimens, and B19-IgM was also positive in all acute phase sera. B19-IgG in most of all sera was kept in peak level up to 216 days after onset. B19-IgM increased rapidly in acute phase and seemed to disappear within one to 5 months after onset. Thirty-seven specimens including 14 obtained at state before infection and 23 controls were completely negative for both B19-IgG and IgM. A new generated B19-antigen specifically discriminated B19-antibodies in 4 epidemics during 25 years from 1968 to 1992 in Japan. The findings allow that the antigen is a useful tool for B19-antibody detection universally.

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It has scarecely been known that the pathogenetic role of mucoid Pseudomonas aeruginosa being colonised on the airway surface in diffuse panbronchiolotis DPB. The role of alginate, a main component of mucoid substance of Pseudomonas aeruginosa was demonstrated in reference to DPB.
1. Clinical Observation
Serum titer of anti-alginate antibody IgG in the group of Pseudomonas-positive DPB was higher than that of the groups of Pseudomonas-negative DPB (p<0.01) and healthy volunteers (p<0.01).
The concentration of immune complex in Pseudomonas-positive DPB group at serum level was also higher than that of the above two groups (p<0.01), and it was well correlated between activity in the patient's symptom and the patient's prognosis.
2. Experimental Observation
In the immunised mice made by free alginate injections, a lymphocyte infiltration around small vessels and small airways in lung was characteristically found at the early stage after the inhalation with mucoid Pseudomonas aeruginosa PT1252. It disappeared 14-15 days after the inhalation. By repeating the inhalation for 6 days, such a lymphocyte infiltration had been persisted and lymphocyte granulomatous change was formed around small airways. The transformation and narrowing of the small airway occurred by lymphocyte granulomatous change. At the same time, some degree of neutrophil infiltration into the airway was also observed. These findings were closely similar to that of human diffuse panbronchiolitis.
3. Conclusive Words
From the above, the pathogenetic role of alginate in mucoid Psuedomonas aerginosa colonised on airway surface in DPB patient will be explained as follows.
Local immunoreaction of antigen and antibody through alginate evolved lymphocyte infiltration around small airway area. A persistant localization of antigen (alginate) makes such an immunoreaction repeat. Consequently, lymphocyte-granulomatous change is formed around the small airway.
On the other hand, a state of excess antigen introduced by long term colonization of mucoid Pseudomonas aeruginosa forms an immune complex in the host side. Neutrophil combined with the immune complex deposited on the airway surface may act in a destructive manner for the lung tissue of DPB patient.

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The distribution of the genes of streptococcal pyrogenic exotoxin (speA, B and C) among the 400 clinical isolates of Streptococcus pyogenes from Japanese pharnyx was detected by the PCR method (Nihonrinsho 50: 326, 1992).
Sixty, 399 and 303 isolates were positive for the speA, B and C genes, respectively. However, only one isolate had none of these genes. Several isolates possessed two or three genes, i.e., both speB & C, speA & B or speA, B & C were found in 258, 30 and 30 isolates, respectively. No isolate possessing both speA & C without speB was found in this study.
We concluded that the PCR method is much more useful for the epidemiological study on streptococcal pyrogenic exotoxins (SPE) of S. pyogenes isolates because of specificity, rapidity and sensitivity of this method compared with the conventional SPE identification method.

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To establish a model where the role of bacterial biofilms in chronic pneumonia with Pseudomonas aeruginosa could be investigated, hydrocortisone-treated guinea pigs were given P. aeruginosa, strain 2126 by inhalation which were used throughout this study, in planktonic form. In these animals, the bacteria were recovered only from the lungs more than 4 weeks after infection. The persistence in bacterial colonization in the lungs coincided with the formation of glanulomatous lesions that surrounded spherical grains consisting of outer shell and inner bacterial colonies. The outer shell of grain was stained with ruthenium red and was presumed to be polyanionic and therefore to be a biofilm-like material. In normal animals without hydrocortisone-treatment, the number of neutrophils recovered from bronchoalveolar lavage fluid increased significantly from 3 hours after infection and subsequently the inhaled bacteria were eliminated from the lungs by day 3 of infection. This early influx of neutrophils into the lungs tended to be suppressed by treatment with hydrocortisone. The formation of grains did not take place in the lungs of normal animals, indicating the significant role of grain-formation in the initiation and the prolongation of bacterial colonization in the lungs.
P. aeruginosa, strain 2126, incubated in saline formed thick biofilms on the surface of teflon piece. Levofloxacin (LVFX), a quinolone antibacterial, exhibited killing activity against the bacteria in in vitro-forming biofilms at MIC. In contrast, gentamicin (GM), an aminoglycosid antibiotic, and ceftazidime (CAZ), a β-lactams antibiotic, showed no such killing activity at MIC. Treatment of this model with oral LVFX achieved complete eradication of the bacteria, whereas subcutaneous injection of GM or CAZ was hardly effective. The pharmacokinetic study on these antibacterials revealed that the doses used in this study were sufficient to obtain the pulmonary levels of these drugs far above MIC even in GM and CAZ. These data indicate that the outer shell of grains, a characteristic finding in the pulmonary lesions of this model, may be one of the forms of pseudomonal biofilms and that this model represents the significant role of biofilm mode of growth of P. aeruginosa in persistence in pulmonary colonization.

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We studied the protective effect of human macrophage colony-stimulating factor (M-CSF) of fungal infection due to systemic aspergillosis in normal mice. We also examined the effect of M-CSF against the activities of mouse peritoneal macrophage which were relating to the phagocytosis, the killing, the production of superoxide after contacting with phorbol myristate acetate and the production of nitric oxide after contacting with interferon-γ in vitro.
M-CSF improved the median survival time and the survival rate of systemic aspergillosis. Combination therapy with M-CSF and amphotericin-B (AMPH-B) showed the therapy with either M-CSF or AMPH-B alone. M-CSF enhanced the activities of phagocytosis and the killing of ingested Candida albicans H and spores of Aspergillus fumigatus K by macrophage. Furthermore, M-CSF promoted the production of superoxide and nitric oxide in macrophage.
These results indicate that M-CSF can enhance the fungicidal activity of macrophages by activation in vivo, thereby preventing the dissemination of fungal infection.

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Recently, it has been suggested that macrolide antibiotics act as immunomodulators. In this study, we evaluated the effect of EM on macrophage function. We used the mouse macrophage cell line, J774.1. The following direct effects of EM on macrophage function were evaluated: chemotaxis to EM, chemokinetic effect by EM, and the effect of EM on macrophage growth. In order to examine the indirect effects of EM on macrophage functions, we preincubated macrophages with several concentrations of EM and then removed the EM. Thereafter, the phagocytosis of beads, cytocidal activity against Candida albicans, and chemotaxis to lipopolysaccharide were evaluated. EM (at the concentration between 0.04 and 0.2μg/ml) directly stimulated macrophage chemotaxis and chemokinesis. In addition, EM dose-dependently stimulated the growth of macrophages. EM pretreatment (for 4 hours at the contrations between 0.04 and 0.2μg/ml) stimulated macrophage phagocytosis, cytocidal activity against Candida albicans, and chemotaxis to lipopolysaccharide. These results suggest that EM has direct and indirect effects on macrophage functions.

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A 47-year-old male with a history of alcohol abuse had a sore throat on June 8, 1994. On June 13, he had swelling and pain on his right fore-arm. He had tense swelling, redness and pain on the right lower abdomen, left upper arm and left lower leg with high fever and noticed erythema and blisters on his back of the right hand on June 18, which gradually expanding to the entire fore-arm. He was admitted to the local hospital on July 2, where he was operated with excision of the skin and drainage for an abdominal subcutaneous abscess and was given three antibiotics and an intravenous immunoglobulin preparation. Although he showed transient hypotension and moderate liver dysfunction, his condition improved day by day under such treatment. He was transferred to our hospital on July 7 because of the unknown etiology. Aspirate from the abscess contained gram-positive cocci in chains, and group A streptococci were isolated. Panipenem/betamipron was used for an antibiotic during roughly two weeks and excision of the skin and drainage for abscess was performed twice. His skin lesions were continued to improve, normalizing peripheral white blood cell counts, serum levels of CRP and the liver function. On July 24, the antibiotic was changed to intravenous ampicillin and administered for 16 daysand amoxicillin was given orally after that, and he was discharged on August 16.
An isolate of the infecting Streptococcus pyogenes produced pyrogenic exotoxin A, B and the serotype was T-3 type.

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In September 1990, a 55-year-old female with erythroleukemia was treated with enocitabine, mitoxantrone, vincristine, and etoposide. Despite prophylaxis of infectious diseases by oral administration of 2, 400 mg/day amphotericin B (AMPH), 600 mg/day ofloxacin, and 1, 500 mg/day kanamycin, pneumonia with refractory pyrexia appeared and developed cystic lesions with air crescent signs thereafter. Finally, the cystic one formed fungus balls. The pneumonia was diagnosed as aspergillus pneumonia by fungus growth in the tissue in the transbronchial lung biopsy specimens and by an elevation of serum anti-Aspergillus antibody.
The patient had continuously been administered with AMPH for 16 days, increasing the drug doses every 2 days. The maximum plasma level of AMPH rose up to 0.78μg/ml, the total amount up to 166 mg. The fungus balls disappeared completely without adverse effects except a transient decrease of plasma potassium level.
Pharmacological studies had been reported that tissue AMPH levels elevated more than twice as much as that of the plasma. Although the maximum plasma level was less than that of MIC for Aspergillus, the lung tissue drug level was suspected to have been maintained higher by continuous drip infusion.
These findings indicate that continuous drip infusion of AMPH is one of the useful treatment for lung aspergillosis.

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We encountered 5 cases of Clostridium difficile enteritis that is rate in infancy. Its clinical symptoms consisted mainly of diarrhea, fever and vomiting. Also progressive abdominal enlargement was characteristically noted. Hematological examination revealed an increase in the number of leukocytes predominant with granulocytes in addition to accelerated erythrocyte sedimentation rate, an incrase in α2 globulin value and high LDH. On the bacteriological examination, detecting bacteria was difficult because of diarrhea being frequent and small in quantity, so bacteria were detected in only 2 out 5 cases. However, reaction to a CD latex agglutination test turned positive in all the cases during the clinical course, which was consistent with changes in the symptoms. Prognosis was good except one cases which in the symptoms. Prognosis was good except one case which had recurrence.
Early diagnosis and early administration of Vancomycin are important in preventing severe complications. To that end, the CD latex agglutination test is considered useful even at present in the domain of pediatrics.

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