Through a year from April, 1999 to March, 2000, 20 samples, which consisted of raw sewage (2), chlorine-treated sewage (2), seawater (10) and naturally grown oysters (6), were collected monthly both from the sewage works at Mihama-ku, Chiba City and at a yacht harbor in Chiba City Bay, Japan. Astrovirus RNA were detected by reverse transcription-polymerase chain reaction (RT-PCR) and was typed by direct sequencing. Astrovirus positive products were detected from 9 samples (raw sewage; 1/2, chlorine-treated sewage; 2/2, seawater; 5/10 and oysters; 1/6) collected in April, 1999. In May, positive products were detected from 4 samples (raw sewage; 2/2 and seawater; 2/10). In June, only 1 positive product was detected from raw sewage. The number of positive samples showed a tendency to decrease and no positive products were detected from samples collected in July, 1999 to January, 2000. After that period, positive products were again detected from 3 samples (raw sewage; 1/2, chlorine-treated sewage; 2/2) collected in February, 2000. In March, the number of positive samples showed the peak and positive products were detected from 12 samples (raw sewage; 2/2, chlorine-treated sewage; 2/2, seawater; 7/10 and oysters: 1/6). Astrovirus positive products detected in April, May, June, July, 1999 and February, 2000 were classified into type 1 or 2 by sequencing, whereas in March, 2000 were type 1, 2, 3, 6 and 7.
The Shigatoxin detection kit based on the immunochromatography system is commercially available. To obtain the identification result rapidly, we devised the improved method (ICG-Imp) replaced to an original method (ICG). Modification provided that Shigatoxins extracted directly from the strains grown on TSI medium without centrifugation. ICG-Imp was compared with ICG, RPLA and PCR. Comparing with RPLA, the sensitivity, specificity, and concordance rate of Shigatoxin 1 showed 77.5, 100 and 90.5%, respectively on ICG, 93.8, 100 and 97.4%, respectively on ICG-Imp, and 100, 99.1 and 99.5%, respectively on PCR. On the other hand, the patterns of Shigatoxin 2 showed 95.3, 100 and 96.3%, respectively on ICG, 100, 100, and 100%, respectively on ICG-Imp, and 100, 100 and 100%, respectively on PCR. The time required from TSI medium to the final result are 24h, 30-60 min, 48h, and 6h, respectively by ICG, ICG-Imp, RPLA, and PCR. It seems that the ICG-Imp is recommended for the identification by means of the accuracy and rapidness.
With the purpose of studying the involvement of physicians to zoonosis, investigation on the consciousness was performed by disseminating a questionnaire to members of Medical Associations of Kobe City (2, 584 members) and Fukuoka City (1, 814 members). From the members in Kobe, 1, 165 replies were collected (answer ratio: 45.1%) and 774 replies from the members in Fukuoka (answer ratio: 42.7%). About 70-80% of all physicians answering the questionnaire stated that they had examined the patients with infectious diseases at a ratio of less than 10% to a total of patients examined. Among them, 70% of the doctors had asked the patients as to whether or not the patients were keeping pet animals or had traveled overseas recently when infectious diseases was suspected. There were 738 doctors (38.1%) (1, 355cases), who had suspected or made definitive diagnosis on 15 diseases of zoonosis as stipulated in the new Infectious Disease Control Law for the recent 5 years. In all, 365 doctors (18.9%) examined the patients who were suspected to have had an infection from pet animals. The causative animals primarily included dogs, cats, and birds such as parakeet, while monkeys, tortoises, etc. were found in several cases. About one half of the physicians felt that the cases of infectious diseases and zoonosis would increase in the future. In the entry of free opinions relating to the effective measures for the prevention of zoonosis, 712 physicians (39.8%) entered 1, 050 proposals. The details of these proposals were: 444 proposals on“administrative measures”, 244 on the need of “education” to citizens, and 201 on the importance of“medical management”. About 80% of the physicians replied that they would cooperate for the secondary investigation. It appears that the physicians place much expectation on adequate measures against zoonosis. Namely, they expect much on the establishment of a reliable network, i. e. the network of administrative authorities-physicians-veterinarians-pet animal suppliers-pet animal keepers.
We clinically analyzed 83 patients with community-acquired pneumonia caused by a mixed infection of polymicrobial agents who we have treated during the past 15 years. A comparative study among three groups; an infectious group with polymicrobial agents (83 cases), an infectious group with monomicrobial agents (335 cases), and an infectious group with unknown agents (599 cases) was performed. The results were as follows; (1) The highest percentage of patients were elderly and bedridden.(2) Striking atypical pneumonic symptoms, including dyspnea, consciousness disturbance, gastrointestinal symptoms and hypotension (shock) were present.(3) Laboratory findings of poor nutritional conditions, including decreases in serum protein, albumin, and cholineesterase, and hypoxia remarkably increased.(4) The prognosis was poor because the mortality rate (15.7%) was higher.(5) There were two polymicrobial agents for 75 patients and three agents for 8 patients. The coupling of polymicrobial agents was most frequent in five patients with Haemophilusinfluenzae+MSSA and five with H. influenzae+respiratory virus. These results suggest that the patients with community-acquired pneumonia caused by a mixed infection of polymicrobial agents had clinical features and causative microorganisms resembling those of elderly patients with community-acquired pneumonia. We recommended that treatment with antibiotics for them was adequate if the treatment resemble that of elderly patients.
Agar media for isolation of enterohaemorrhagic Escherichia coli (EHEC) have been developed primarily for E. coli O157, because this bacterium has most frequently caused EHEC infection. However, there have been few studies for isolation of other serotypes of EHEC, and media appropriate for isolation of such organisms, especially from food samples, are not yet available. Among such serotypes, E. coli O26 has often been isolated from clinical specimens from patients and animals, but not from food samples in outbreaks, because of lack of an appropriate method for isolation. In this study, we tried to develop anew chromogenic agar medium for selective isolation of E. coli O26 using the characteristicsof E. coli O26. Fifteen strains of E. coli O26, 11 strains of E. coli O157 and 36 strains of other serotypes E. coli were tested for fermentation of rhamnose, cellobiose, dulcitol, salicin, raffinose, sorbitol, sucrose, lactose, mannitol, arabinose, maltose, xylose and glucose. Rhamnose was fermented by all E. coli strains except for E. coli O26. The other substrates were not effective for differentiating E. coli O26 from the other strains of E. coli. Thus the medium containing rhamnose and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, which is a substrate of β-galactosidase specific to coliforms, produced acolor of E. coli O26 colonies different from colors of the other bacteria. Furthermore, cefixime and sodium tellulite were added to the composition of the medium for gaining higher selectivity.
The ability of the AccuProbe (Kyokuto) to identify mycobacteria directly from the positive cultures in an automatic detector for mycobacteria (BACTEC MGIT960, Becton Dickinson) was evaluated. Sputum samples were collected from patients with suspected mycobacteriosis between February and April 1999 and conventionally incubated in MGIT960 (37°C for 42 days). The MGIT-positive cultures were successively incubated for several days and were directly identified with the AccuProbe. Monomycobacterial strains were detected from 93 (93.9%) of the 99 sputum samples and polymycobacterial strains were detected from 6 sputum samples (6.1%). Viable cell counts in the positive cultures in MGIT960 were determined using Middlebrook 7H10 agar. The cell counts of Mycobacterium tuberculosis and Mycobacterium avium-intracellulare complex (MAC) varied greatly among the individual samples; the former, 3.8×102-2.5×106cfu/ml and the latter 1.5×103-1.9×108cfu/ml. The great differences in the cell counts were observed among these samples. Although the cultures in MGIT were estimated as positive, the early stage of bacterial growth might be used as the samples for the cell counting. By this method, 96 of the total 101 cases were successfully identified as follows: M. tuberculosis 57 cases (56.4%) and MAC 39 cases (38.6%). In conclusion, the present results indicate that the direct identification of mycobacteria from the positive cultures in MGIT960 using AccuProbe is useful for a rapid diagnosis in microbiological testing for mycobacteriosis which has tended to increase in recent years.
Sucrose-negative colonies were isolated on TCBS agar plate from otorrhea specimens from otitismedia patient in the Adult Diseases Examination Center, Hirosaki Medical Association. The isolatewas identified as Vibrio vulnificus by nested PCR method which amplify V. vulnificus-specific sequencesdirected against 23S rRNA genes. The PCR method was applied to identify 6 strains originallyisolated from septicemia patients in Kurashiki Central Hospital and formerly identified as V.vulnificus by phenotypic characteristics. When examined by the API20E system, the above PCRconfirmed-V. vulnificus isolates were correctly identified as V. vulnificus with % ID 99.8, though thisgave 3 different profiles. Cytotoxin-hemolysin gene was detected in otorrhea strain as well as septicemiastrains by PCR method. The above results suggest that PCR method targeted cytotoxinhemolysingene is suitable for rapid and accurate identification of the isolate, because the result is obtainedin less than 4h. To our knowledge this is the first report on the V. vulnificus infection in Aomori Prefecture andthe isolation from otorrhea.
During the 20-year period between 1979-1998, a total of 4, 176 strains of hemolytic streptococcihave been isolated from 20, 118 healthy primary school children and little children in Tokyo Metropolitan (Tokubetsuku, Tama and Tosho). Culture of throat swabs every November and the following February during the 20-year period were made and serological grouping and typing for isolates weredone by T agglutination method. The results were as follows. 1) Serological group of hemolytic streptococci isolated from children were 3, 188 strains (76.3%) for isolates of group A out of total strains of 4, 176, 569 strains (13.6%) for isolates group B, 63 strains (1.5%) for isolates of group C and 356 strains (8.5%) for isolates of group G. 2) The most dominant was T12 during 1979-1998, and other relatively frequent serotypeswere T28, T1, T4, T6 in that order. These ranks of and the main epidemic serotypes showed a similartrend in the 3 areas. 3) The isolation rates of group A streptococci were 15.9% in Tokubetsuku, 17.1% in Tama and14.9% in Tosho. The average of 3 areas were 15.8%. 4) The epidemic cases seemed to be caused by group A streptococci were 20 cases, their isolatedserotype were 7 cases by T28, 5cases by T12, 4 cases by T6, 2 cases by T4, each 1 case by Tiand T25. 5) A total of 2, 927 strains of group A streptococci were examined for drug sensitivity. Allstrains were sensitive to 13-lactam group of antibiotics (benzylpenicillin and cephaloridine). Resistant (MIC≥25μg/ml) to TC, CP and EM etc. were 740 strains (25.3%) in this study. The incidence of resistant strains were to TC 493 strains (66.6%) out of 740 strains, 81 strains (10.9%) for TC·CP, 72 strains (9.7%) for EM and 66 strains (8.9%) for TC·CP·EM·OL·LCM. TC resistant strains have not varied much through the whole period, but CP and EM resistantstrains were very variable by year. Many resistant strains to TC were T4, to EM and multiple drug resistant were T12. 6) The rates of isolates of the same type of group A streptococci in school child individual duringfor the tests taken twice a year were 12.3%, indicating group A streptococci, according tothe durationof the carrier state, seems to be a short period.
It is important to develop a sensitive assay method for evaluation of immunomodulating candidatesincluding granulocyte-colony stimulating factor (G-CSF) which may augment host defense functionagainst opportunistic infections. Effects of CSFs on anti-Candida activity of bone marrow cells (BMC) obtained from normal or cyclophosphamide-treated immunocompromised mice (CY-mice) were investigated. When BMC from CY-mice were preincubated for 24 hours in the presense ofmore than 0.005ng/ml of G-CSF, anti-Candida activity was enhanced at 160 of effector (BMC) to target (C. albicans) ratio, whereas anti-Candida activity of BMC from CY-untreated mice was notchanged at concentrations less than only by more than 0.5ng/ml of G-CSF. Similar enhancement ofactivity was also observed in the presense of more than 0.05ng/ml of granulocyte-macrophage colonystimulating factor (GM-CSF) in this assay using BMC from CY-mice. These findings suggest thatthis in vitro assay method can evaluate the activity of G-CSF and GM-CSF to augment leukocyte'santi-Candida activity.
From October 1998 to April 1999, 72 stool specimens from patients of acute gastroenteritis werecollected in a pediatric clinic in Urawa city, Japan and were examined for the detection of SRSV byelectron microscopy and reverse transcription-polymerase chain reaction (RT-PCR). Forty-eight (67%) out of 72 were SRSV positives by both tests. In this season, a prevalence of SRSV peaked in December1998. Among reported clinical symptoms, vomiting was reported more frequently than theother symptoms, such as diarrhea and fever. Furthermore, the diarrhea was intense among childrenunder 1 year old. For the detection of SRSV by RT-PCR, we used two kinds of primer sets (MR3/MR4 and P1/P2/P3). The P1/P2/P3 primer was most sensitive for the detection of SRSV.
A 15-year-old girl, high school student, became febrile (38-39°C) with chills, more throat andcough on April 20, 1994. Until the onset, she was healthy and she had been camping with her classmatesin a wooded mountainous area in Oku-etsu, Fukui Prefecture. She consulted a local clinic on.April 21 and bacampicillin was initially administered and then changed to cefaclor on April 23. However, high body temperature continued and a maclopapular rash appeared on her face on April 24and gradually spread to her anterior chest and back. Blood examination showed a WBC count of2, 200/μl and she was admitted to our hospital on April 25. On admission, peripheral blood data showed leukocytopenia (WBC 2, 300/μl) with 5% atypicallymphocytes. Titers of anti-Rickettsia typhi serum antibodies (IgM, -G) were elevated (1: 80, 1: 640) and she was diagnosed as having murine typhus. On the second hospital day, 200 mg of minocycline (MINO) was administered per os and her body temperature fell to within the normal limits on thethird hospital day. On the 7th hospital day, the skin rash disappeared and she was discharged. Altogether, 320 high school students went camping with this patient. Among them, approximately 30 studentshad similar symptoms and signs as this case and had been diagnosed suspected viral infection.Twelve students of the 30 were admitted to other hospitals. It was considered that this case was partof an outbreak of murine typhus in the Oku-etsu area, Fukui Prefecture, but no further investigationwas performed. Murine typhus is usually a benign disease that is controllable by the administrationof MINO. In rare cases, infection can worsen to multiorganic failure, severe complications have beenreported in 1-4% of cases, and death has been reported in less than 3%. Recently, it has also beenreported that MINO not only has an antibiotic effect, but also play acts as a cytokine modulator in patientswith rickettsial infection. Thus, in febrile patients in whom uncommon Rickettsia infection issuspected, serological test for murine typhus should be examined and the immediate administrationof MINO is important.
Mortality of meningococcal septicemia remains high in spite of the improvement of antibioticstreatment and critical care medicine. A 23-year-old male, who had been well until a day earlier, wasadmitted to the hospital because of a high-grade fever and headache. On the second hospital day, hewas still febrile, and it was confirmed that he had disseminated intravascular coagulation. There wasno purpuric skin lesion, and a lumbar puncture revealed no abnormality. The condition was complicatedby a splenic infarction on the second hospital day, and he suffered a pulmonary infarction onthe 8th hospital day. The blood culture was positive for Neisseria meningitidis, making the diagnosis meningococcalsepticemia. He was successfully treated with antibiotics and intensive care. Although meningococcocemia in adults is relatively rare in Japan, the disease mortality is stillhigh even in the modern era. Then, once the diagnosis is suspected, it is essential to keep in mind thatmeningococcal infection requires early recognition of the disease process, prompt initiation of adequateantiinfectious therapy and intensive treatment of multiorgan failure.