In Western countries, prevention of influenza has been a major public health concern, promoting the vaccination program to high-risk individuals including the elderly.On the other hand, in Japan, there has been no systematic approach to such a selective vaccination on the social basis. This is due to the deep-rooted skepticism on vaccine efficacy. A number of epidemiologic studies have so far reported conflicting results in this country. We investigated the principles and methods of influenza epidemiology focusing on the field evaluation of vaccine efficacy.
General and methodologic problems in vaccine field trials with naturally occurring influenza include; unpredictability of the time of its occurrence; antigenic differences between the vaccine strains and epidemic viruses; preexistence of already-immuned individuals; indirect effect of hard immunity by vaccination on nonvaccinees; possible difference in the virus exposure between compared groups, particularly when epidemic scale is small; possible misdiagnosis of cases, if not laboratory-confirmed.
To copewiththese, the following measures are essential in conducting epidemiologic study on influenza.
1) Tnfluenza epidemics show differential occurrence by time and place. Therefore, much attention should be pain when analyzing the pooled data obtained from various study samples at different locations.
2) It should be the first step of a research to consider whether the outbreak of acute respiratory illnesses observed among subjects is caused by influenza virus exposures.
3) In general, faillings to detect vaccine efficacy are attributable to the dilution of outcome with noninfluenzal illnesses; cases defined by clinical symptoms include substantial number of acute respiratory illnesses other than influenza. To minimize this nondifferential misclassification, three methods are thought to be important; confining observation period during the peak epidemic; applying a strict criteria to measure the outcome, preferably with laboratory examination; and conducting the study in the season with large-scale epidemic.
4) It is also important to take into account the preexistence of already-immuned individuals. “Antibody efficacy” is a keen index to assess vaccine effectiveness in this instance.
Besides, further research is required to clarify the individual characteristics related to influenza attack, which affect the validity of analytic epidemiologic study on vaccine efficacy by yielding bias or confounding effect.

View full abstract

The role of clarithromycin (CAM) in biofilm formation has recently been reported. Inhibition of the production or promotion of the dissolution of the glycocalyx, a major component of biofilm, has been implicated in its mechanism of action. However, the details remain unclear. We used an experimental model of complicated urinary bladder infection and automated simulation of the variations in urinary antimicrobial concentration to study the efficacy of CMA in inhibiting biofilm formation and obtained the following results.
1) Priro to biofilm formation, Pseudomonas aeruginosa (P. aeruginosa) was exposed to ciprofloxacin (CPFX, MIC: 8μg/ml), which was active against the organism, at a dose of 200 mg t. i. d. for 7 days. The bacteria were apparently eradicated from the culture medium in the experimental model of bladder infection (model bladder) after 32 hours. However, when the medium was changed to eliminate the antimicrobial agent on Day 7, bacterial regrowth was initiated after 4 hours. Scanning electron microscopy demonstrated sequential biofilm formation on the surface of glass beads in the model bladder diverticulumn, suggesting inside the biofilm were a source of regrowth.
2) Prior to biofilm formation, P. aeruginosa was also exposed to CAM alone, which has no antimicrobial activity against the organism (MIC:> 128μg/ml) at a dose of 200 mg t. i. d. for 7 days. In this situation, CAM was not active against P. aeruginosa and the bactericidal concentration in the model bladder did not decrease markedly, reaching the initial level (10⁷ CFU/ml) within 48 hours. However, although numerous bacteria were attached to the glass beads in the diverticulum, no biofilm was formed.
3) Exposure to a combination of CPFX and CAM (each at 200 mg t. i. d. for 7 days) resulted in the eradication of bacteria from the model bladder at 32 hours, and no bacterial regrowth was demonstrated after the medium was exchanged on Day 7. In addition, no biofilm was formed and the bacteria did not become attached to the glass beads.
4) The content of alginate, a major component of P. aeruginosa biofilm, was measured per 5 glass beads on Day 3, 5, and 7 after starting drug administration. The alginate content increased with time when CPFX was given alone at a dose of 200 mg t. i. d. In contrast, the alginate content increased slightly on Day 3, but decreased to below the detection limit on Days 5 and 7 when CAM was given alone at a dose of 200 mg t. i. d. In addition, the alginate level was also below the detection limit on Day 7 when a combination of CPFX and CAM was given (each at 200 mg t. i. d.).
5) Therefore, the inhibition of glycocalyx production may be implicated in the mechanism by which CAM prevents biofilm formation.

View full abstract

Immunoglobulin M and G antibody responses in mice experimentally infected with Toxoplasma gondii (S-273) and the reaction patterns with T. gondii (RH) tachyzoite antigens were studied on various post-infection days (PIDs) (2nd to 36th PIDs) using a commercially available IgM and IgG enzyme linked immunosorbent assay (ELISA) test systems and Western blotting (WB) technique. IgM antibody in ELISA test appeared to be positive on 12th PID (absorbance 0.764) and reached its peak level on 16th PID (absorbance 1.338) showing a slow decline thereafter with an absorbance of 0.800 even on 36th PID. Positivity of IgM was confirmed by WB except for 36th PID. IgG appeared on 16th PID with an absorbance of 0.248 and showed a steady increasing tendency even on 36th PID (absorbance 1.747). However, IgG positivity on WB was observed only on 29th PID and afterwards. On Western blots, both IgM and IgG showed interesting antigen recognition patterns on various PIDs. At the most IgM recognised seven antigens of 14kDa to 53kDa while IgG recognised eight antigens of l7kDa to 53kDa. Major antigens recognised by IgM were of 53kDa and 21kDa while the major band recognised by IgG was of l9kDa. The major bands, however, showed variability in their consistency during various PIDs. All the antigens recognised by IgM and IgG were not identical.

View full abstract

Although erythromycin estolate has been fully assessed for pertussis treatment, the evaluation of erythromycin ethylsuccinate and stearate, the main erythromycin preparations used in Japan and the US, is inadequate. We evaluated these preparations to establish an appropriate treatment for pertussis according to age. Sixty-six patients with culture-confirmed pertussis were treated with erythromycin administered at a dosage of 40-50 mg/kg/day (maximum, 1.2g/day). Negative culture was obtained in 39%(15/38) of patients aged 0-2 years within one week and in 71%(27/38) within two weeks, in 78%(7/9) of those aged 3-15 years within one week and in 100%(9/9) within two weeks. All 12 adult patients had a negative culture within one week.
The efficacy of erythromycin for the eradication of B. pertussis was significantly lower in children aged 0-2 years than in older children. In conclusion, it is desirable to administer erythromycin for three weeks to children aged 0-2 years, two weeks to those aged 3-15 years and one week to adults.

View full abstract

I have constructed the genomic library of M. leprae Thai 53 strain, and cloned the α antigen like protein gene by plaque hybridization method by using M. leprae α antigenDNA fragment as probe which was characterized in the previous study, I have termed it as α2 antigen gene. The α2 antigen gene has been characterized by sequencing. By comparing the deduced amino acid sequence of α and α2 antigen with 85 complex antigen of other mycobacteria. Ihave found the higher homology between α2 antigen and 85A antigen and between a antigen and 85B antigen.
We have constructed the over expression system of M. leprae α and α2 antigengene in E. coli using vector pMALc-RI. Recombinant α and α2 antigen has been purified byamylose column chromatography at the purity of more than 95%. More than 6 mg and more than 10 mg of recombinant α and α2 antigen has been obtained from 200 ml of liquid culture, respectively.
ELISA tests have been performed with the sera of leprosy patient and healthy control against the recombinant α and α2 antigens. The antibody titers in sera of leprosy patient against the two kinds of antigens were all much higher than healthy controls. The antibody titer against the α2 antigen was higher than that against α antigen. Recombinant α and α2 antigens in this study could be used as a new specific antigen for serodiagnosis of leprosy.

View full abstract

In order to investigate penicillin resistance (Pc^r) in Streptococcus pneumoniae from clinical sources in Japan, a total 1, 127 strains of S. pneumoniae was collected at random from 36 insititutions participating to “Working group for Pc^r S. pneumoniae” around the country in 1993-1994. These strains were isolated more frequently from sputum (38.2%), throat (31.4%), nasal discharge (16.4%), and otorrhea (5.7%). A small number of isolates from blood (19 strains; 1.8%), cerebrospinal fluid (11 strains; 1.0%), and pleural fluid (2 strains; 0.2%) were included respectively. Patients from whom S. pneumoniae was isolated have mostly been associated with children≤ 12 years of age and adults 60≤years olds. These isolates were tested for susceptibility to penicillin G, ampicillin, oxacillin, cefixime, cefdinir, imipenem, panipenem, erythromycin, clindamycin, minocycline, and vancomycin by an agar dilution method using Mueller Hinton agar supplemented with 10% sheep blood. Strains with the MICs≥0.125μg/ml for penicillin G were defined as a Pc resistance. Of the 1, 127 strains, 471 strains (41.8%) were identified as a Pc resistance. Pc^r S. pneumoniae were almost resistant to other β-lactams, including ampicillin, oxacillin, ceftizoxime, cefixime, cefdinir. Although, the MICs of imipenem and panipenem ragned from 0.004-2.0μg/ml with 2 peaks distributions, these antibiotics inhibited the growth of most of Pc^r S. pneumoniae at the lowest concentrations of ≤0.5μg/ml. Only vancomycin resistant strain was not detected in these isolates. Most of the Pc^r strains were simultaneously resistant to macrolides and minocycline. Further more, isolation frequencies of Pc^r S. pneumoniae in west Japan, were relatively high compared with those of east Japan.

View full abstract

We isolated Salmonella choleraesuis subsp. choleraesuis serovar. Enteritidis (S. Enteritidis) from a mass of cases which broke out in September, 1991 in Kumamoto City. The isolates were shown to hold plasmids on which a tetracycline (TC)-resistant gene was located. The plasmid, about 9 kb in size, was capable of expressing the gene in Escherichia coli, unstable in S. Enteritidis and Escherichia coli. Thus in order to investigate the creeping prevalence of the TC-resistant plasmid in Kumamoto City and its spreading in other areas, the strains, a total of 41, including 37 isolates from sporadic cases with diarrhea in Kumamoto City, 3 isolates from 3 cases of food poisoning in Kitakyushu, Fukuoka and Chiba, and one strain isolated from egg solution in Kumamoto City, were examined by the hybridization test with this plasmid as a probe, as well as for their drug sensitivity and plasmid profile.
As a result, 28 strains of 37 tested from the sporadic cases, the strains from food poisoning in Kitakyushu, Chiba and the one from egg solution, all harbored the same 9 kb TC-resistantplasmid.
From the above observations, it was assured that, at the time of investigation, July to September in 1992, the prevalence of S. Enteritidis in Kumamoto City was Predominated by its strain carrying the TC-resistant plasmid. Furthermore, considering the fact that the strain harboring this plasmid was also found in the food poisoning in Kitakyushu and Chiba, it was suggested that spreading of this strain was not restricted to localities and was also associated with Chicken eggs according to its detection from the egg solution.

View full abstract

The pattern of distribution of bacteria, Mycoplasma pneumoniae and virus isolated from the same specimen recovered from the throat swab or the sputum of 479 patients with respiratory infections who were seen in six private clinics in Sendai City of Japan during the period from October to November in 1992 (period I) and from January to February in 1993 (period II) was documented. Of the 479 patients, 234 had acute pharyngitis, 145 had acute bronchitis, 96 had influenza, 21 had acute tonsillitis, 5 had acute pneumonia and 9 had other respiratory infections. One hundred (42.4%) strains of potential pathogen and one strain of M. pneumoniae were recovered from 236 cases in period I, and 66 (27.2%) strains of potential pathogen, one strain of M. pneumonae and 73 strains of Influenza virus (30.0%: 43 of type A Hong-Kong and 30 of type B) from 243 cases in period II. Of the 166 strains, major isolates were Staphylococcus aureus (56 strains), Streptococcus pneumoniae (12 strains), Streptococcus pyogenes (15 strains), Haemophilus influenzae (17 strains), Esherichia coli (4 strains), Klebsiella spp. (35 strains), Pseudomonas aeruginosa (4 strains) and Acinetobacter spp. (23 strains). Only one strain of S. aureus was resistant to methicillin (MIC: 50μg/ml). None of S. pneumoniae was resistant to 1 μg/ml of ampicillin. Ciprof loxacin was administered to 113 cases and roxythromycin to 220 cases by doctors in charge. Efficacy rate in period I and period II was 93.7% and 89.4% for ciproxacin, and 82.4% and 88.4% for roxythromycin. Roxythromycin was effective even in patients in whom Influenza virus was detected in period II. The pattern of distribution of pathogenic bacteria in primary care clinics was similar to that in intensive-care oriented clinics i.e. medical schoolaffiliated hospitals. On the other hand, the incidence of resistant strains of bacteria in primary care clinics was low and different from that in intensive-care oriented clinics.

View full abstract

To evaluate the cause of the vulnerability to infections in the elderly, the ability of neutrophil to generate reactive oxygen species was assessed by a luminol-dependent chemiluminescence (CL) assay after stimulation with non-opsonized zymosan, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Candida albicans and lumispheres in elderly patients aged 70 to 93 years. The integrated CL for 20 minutes of whole blood and neutrophils induced by zymosan in the elderly was significantly lower than that in healthy young adults, and the integrated CL of neutrophils induced by lumispheres was also significantly lower in the elderly aged 80 years and over. When bacterial infection occurred in the elderly, the levels of CL were elevated and decreased in the convalescence. This response is proper for host-defense mechanism against infection. However, whole blood CL response was not fully activated in any patients of the elderly during bacterial infection. In these cases lower white blood cell counts, lower neutrophil counts, or the decreased level of the serum total protein, albumin, total cholesterol or cholinesterase were observed. Relationship between malnutrition and the ability of neutrophil to generate reactive oxygen species was suggested. Furthermore, I evaluated the priming effect of lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNF-α) on whole blood CL. The CI, responses stimulated with non-opsonized zymosan or P. aeruginosa were enhanced by pretreatment with TNF-α and LPS in healthy young adults. On the other hand, no significant priming effect was observed when blood from elderly patients were incubated with each primer.
These findings suggest that the impairment in the generation of reactive oxygen species of the neutrophils and the decrease in reactivity to LPS and TNF-α that activate neutrophils at the site of infection and potentiate host defense against invading bacteria, may contribute to susceptibility to infection in the elderly.

View full abstract

Mycoplasma genitalium causes urethritis in non-human primates, but studies on its pathogenicity in man have been hampered by the difficulty in isolating this oragnism in culture. We have used a specific polymerase chain reaction to examine the role of M. genitalium in nongonococcal urethritis (NGU). Oligonucleotide primers were used to amplify a 281 bp of 140-KDa adhesin gene of M. genitalium. A characteristic PCR product was amplified, when M. genitalium DNA was template for the PCR. No amplified product was detected in Mycoplasma pneumoniae DNA, Mycoplasma hominis DNA or other bacterial DNAs. M. genitalium DNA was detected in urethral swabs from 17 (14.9%) of 114 men with NGU. Three (9.1%) of the 33 men with Chlamydia-positive NGU and 14 (17.3%) of the 81 with Chlamydia-negative NGU were positive for M. genitalium DNA, but 29 men without urethritis were negative. The prevalence of M. genitalium in NGU and in Chlamydia-negative NGU was significantly higher than that in the normal control. These findings suggest that M. genitalium would be a cause of NGU.

View full abstract

Studies were conducted to elucidate the immune cell response at infectionsites by performing immunostaining of immune cells with a monoclonal antibody in an experimental Escherichia coli (E. coli) mouse uterine infection model.
1. The incidence of uterine infection by E. coli decreased with the the passage of time: 4/4 on Day 1, 4/6 on Day 3, 2/6 on Day 7, and 1/6 on each of Days 14 and 21. It was surmised that clearlance of the bacteria from the infection sites was being carried outby immune cells.
2. Beginning from infection Day 1, the infected uterine tissue was observed to undergo a moderate degree of invasion by neutrophils, macrophages, CD4⁺ T cells, CD8⁺ T cells and IgA⁺ B cells. Then, beginning from infection Day 3, there was a mild degree ofinvasion of the infected uterine tissue by IgM⁺ B cells and IgG⁺ B cells. The number of neutrophils in the tissue decreased beginning from infection Day 14, but the degree of invasion of the infected tissue by the other kinds of immune cells remained almost constant through infection Day 21.
3. A comparison was made of the immune responses to local infection by E.coli, and Chlamydia trachomatis (C. trachomatis) , an intracellular parasite. It wasfound that the invasion of the infection site by immune cells occurred earlier in the case of E. coli infection than C. trachomatis infection. In addition, the C. trachomatis infection site wasobserved to contain greater numbers of macrophages and CD8⁺ T cells play important roles in the immune defense at sites of infection by C. trachomatis.

View full abstract

A clinical study was conducted on fleroxacin (FLRX) in 143 patients and carriers with infectious enteritis (shigellosis, Salmonella enteritis, Campylobacter enteritis, pathogenic Escherichia coli enteritis, Vibrio parahaemolyticus enteritis, cholera, multiple bacterial infections, pathogen-negative enteritis). Furtheremore, its antibacterial activity against clinical isolates, fecal concentration and effect on fecal microflora were conducted. FLRX was administered orally in doses of 200 mg once a day (200 mg group) or 300 mg once a day (300 mg group) for 3 days to cholera, for 7 days to Salmonella enteritis and for 5 days to the other infectious enteritis.
The clinical efficacy rates were 100% in both the 200 mg and 300 mg groups.
The bacteriological efficacy rates were 100% against Shigella spp., Salmonella spp., pathogenic E. coli, V. parahaemolyticus and V. cholerae O1, and 63.6% against Campylobacter spp. in the 200 mg group. The rates of the 300 mg group were 93.3% against Shigella spp., and 100% against Campylobacter spp. and pathogenic E. coli.
As adverse effects, skin rash was observed in 1 case each in both groups (1.1%, 2.1%). Abnormal laboratory findings consisted of 1 case of increased eosinophils and 1 case of elevated GOT and GPT levels in the 200 mg group (2.8%), and 1 case of elevated GPT in the 300 mg group (2.9%).
The clinical usefulness rates were 92.9% and 93.3% in the 200 mg and 300 mg groups, respectively.
Antibacterial activity was somewhat inferior to that of ciprofloxacin and equal to or better than that of norfloxacin, demonstrating MIC₉₀ values against Shigella spp., Salmonella spp., pathogenic E. coli, V. parahaemolyticus and Campylobacter spp. of 0.1, 0.2, 0.1, 0.2 and 0.78μg/ml, respectively.
Peak fecal concentrations of the drug were 49.0μg/g and 274.4μg/g in the 200 mg group, and 43.3μg/g and below the detection limit (5.0μg/g) in the 300 mg group.
With respect to fecal microflora (4 cases), a decrease in Enterobacteriaceae was observed in 3 cases during dosing. But this change showed a tendency to recover after completion of dosing. No effects were observed on anaerobic bacteria.

View full abstract

Antibacterial activity of fleroxacin (FLRX), a new quinolone antimicrobial, against 36 strains of Shigella app., 14 strains of Salmonella spp., 11 strains of Escherichia coli, 9 strains of Vibrio spp. (including 2 strains of V. cholerae O1), 14 strains of Campylobacter jejuni/coli, 3 strains of Aeromonas spp. and 1 strain of Plesiomonas shigelloides isolated from infectious enteritis patients in this study was determined. Its activity was compared with that of ciprofloxacin (CPFX), norfloxacin (NFLX) and nalidixic acid (NA). The MIC₉₀ values of FLRX were 0.1μg/ml against Shigella spp. and E. coli, 0.2μg/ml against Salmonella spp. and Vibrio spp., and 12.5μg/ml against C. jejuni/coli MIC₉₀ of FLRX was comparable to that of CPFX and NFLX against Vibrio spp. Against other species, MIC₉₀ of FLRX were 2-to 4-fold higher than those of CPFX, whereas equal to or 2-fold lower than NFLX. FLRX demonstrated excellent activity against an NA-resistant (MIC:>100μg/ml) isolate of E. coli, with MIC 0.78μg/ml. FLRX showed 8-fold higher activity than NA against other stratains.
The antibacterial activity of FLRX was compared with that of NA against stocked strains (clinical isolates from August 1989 to February 1991), consisting of 11 strains of Shigella spp., 10 strains of Salmonella spp., 8 strains of E. coli, 10 strains of V. cholerae O1, 10 strains of V. parahaemolyticus and 14 strains of C. jejuni/coll. MICs of FLRX were 0.78 and 12.5-25μg/ml against Shigella spp. and C. jejuni/ coli that showed resistance of NA (MIC:>100μg/ml), respectively.
Based on the above, although the absolute MICs are low against E. coli and shigella spp., a value of 0.78μg/ml for FLRX suggested that such strains should be considered to be resistant.

View full abstract

An 18-year-old female was given prednisolone and azathioprine for treatment of systemic lups erythematosus. She was admitted to Fukuoka University Hospital because of headache and vomiting. Examinations revealed she was suffering from cryptococcal meningitis.
Fluconazole (FLCZ) 400 mg a day was administrated for therapy. Her general condition improved and the serum level of cryptococcal antigen decreased one month after therapy. This therapy resulted in abatement of subjected symptoms and sterilization of the cerebral spinal fluid. This case suggested that therapy with FLCZ alone is useful for patients with cryptococcal meningitis, therefore the standard therapy with amphotericin B (AMPH) or the combination.

View full abstract

This paper reports a case of TSS complicated with SSSS in an adult with liver cirrhosis.
A 52-year-old male, heavy drinker, was referred to our clinic complaining lumbago and painful swelling of the right arm. The patient had peeling of the skin over the hips, knees and elbows with positive Nikolsky's sign. The patient was in a state of shock on admission. Pyrexia persisted for 4 days and finally the body temperature rose up to 39 °C. The laboratory studies revealed hypoxia, DIC and multiple organ failure, and these became progressively worse. He died 4 days after admission.
According to the criteria, he was diagnosed as TSS, and TSST-1 was detected from his serum. Staphylococcus aureus, coagulase type V was cultured both from the blood and from the wound of his right middle finger. This isolated strain did not produce TSST-1. The skin specimen at autopsy showed that the cleavage plane lied at the subcorneal region and close to the granular layer, with specific changes caused by exfoliative toxin. It was compatible to the exfoliation which was caused by exfoliative toxin produced from the S. aureus coagulase type V. The autopsy also revealed alcohol liver injury, liver cirrhosis and multiple organ failure due to shock state.
SSSS is rare in adults, to our knowledge this is the first reported case of TTS complicated with SSSS.

View full abstract

A case of fulminant group A streptococcal infection occurring in a 6-year-old Japanese child is reported. She was accompanied by massive pulmonary hemorrhage and subsequent asphyxia. She initially had pharyngalgia with fever. The cephalosporin antibiotic was given orally for 3 days. Three days after that recurrence of fever and pharyngalgia was noted. Twelve hours later tachypnea and a sudden onset of hemoptysis was noted. She manifested DOA (dead on arrival) and died in the emergency room. Autopsy revealed the presence of numerous cocci in the vessels and massive pulmonary hemorrhage.
Streptococcus Pyogenes was isolated from the blood. The serotype of this group A streptococcal organism was typed as M4, T4, which produces exotoxin type B and C, which was sensitive to the penicillins.

View full abstract

A 64-year-old male was admitted to our division because of fever.
After admission, the patient was given β-lactam antibiotics intravenously because he had no eruption and eschar. However, the fever continued, and he became unconsciousness and DIC appeared. We diagnosed the patient as Tsutsugamushi disease from indirect fluorescent antibody technique. Minocycline was excellently effective.
Several reports of Tsutsugamushi disease without eruption have been given, so we must always be careful of Tsutsugamushi disease

View full abstract