Rinsho Biseibutsu Jinsoku Shindan Kenkyukai shi = JARMAM : Journal of the Association for Rapid Method and Automation in Microbiology最新文献

The serotyping of staphylocoagulase is widely used in Japan. However, the conventional immunoassay based on neutralization of the antisera is so laborious and time-consuming that it is not widely used in the other countries. In order to overcome these drawbacks we developed a novel staphylocoagulase serotyping method based on a microplate format using polystyrene latex particles. Addition of latex particles promotes the formation of fibrin complexes, which represents a more rapidly and easily detected endpoint. For 83 strains, 90% were classified into serotypes within 3 h, and there was no discrepancy in the results between our method and the conventional method. These results indicate that the present microplate method is rapid, simple, and interpretable.

Because the detection of bacteremia is an important indication for subsequent treatment and prognosis, blood culture-positive results should be reported as soon as possible. Here, we present our attempts to promptly report the results of blood culture-positive samples and successive support systems for medical examinations. The BacT/ALERT system is used for culturing blood in our hospital. If a positive blood culture is obtained, medical technologists at the microbiological laboratory of our university hospital immediately contact the corresponding ward regarding the results of Gram staining and simultaneously inform the infectious disease physicians at the Department of Infection Control and Prevention. By using the positive blood cultures obtained from January 2003 to December 2004 (the period during which only day shifts on weekdays was supported), we determined the time required from blood specimen collection to obtaining positive samples from the culture system. When the samples were inserted into the system on the day following blood collection or later, the average time required was 48 hours. On the other hand, when the samples were inserted into the system on the day of blood collection, the average time required was 33 hours. After consideration of the importance of blood cultures, the examination system at the microbiological laboratory was changed to support day shifts every day in May 2008. The time taken from blood specimen collection for blood cultures to obtaining positive samples from the system was 40 hours on an average when day shifts were supported only on weekdays (during the above mentioned period), whereas the average time required was 31 hours when day shifts were supported every day (from May to June 2008). Since January 2002, the Department of Infection Control and Prevention have supported medical examinations for infectious diseases, including blood culture-positive cases. Compared between before and after the establishment of the support system (in 2001 and 2007), the 30-day mortality rates from bloodstream infections with Staphylococcus aureus and Candida spp. have improved from 30% to 17% and 56% to 23%, respectively. As indicated in our results, it is important to immediately deliver blood culture samples to the laboratory, start blood cultures (insert into the system), promptly report the results of Gram staining after the positive samples are obtained, and administer suitable antibiotic treatment.

Diarrhea caused by the Escherichia coli with held adhesion came to attention. We performed an adhesion-related gene and relation of diarrhea. Subjects were 77 outpatients with diarrhea from June 2003 to December 2005. A total of 102 E. coli strains randomly isolated from stool specimens. All the toxigenic examinations were negative, and there were not the relations. Adhesion-related gene were 10 strains found. As for the contents, astA was 5 strains, 2 strains of aggR, 2 strains of eaeA, 1 strain of eaeA plus astA. Of these, we were able to classify 5 strains in serological typing, but remain 5 strains did not typing. Only one strain of O157 was VT positive. There is not it with causative E. coli of diarrhea even if serological typing is negative. Therefore it was thought that an adhesion-related gene test was important.

Minimum inhibitory concentrations (MICs) of vancomycin (VCM) and teicoplanin (TEIC) were measured using a novel susceptibility test based on the chemiluminescence assay method (CA) (Rapid-Lumi Eiken; Eiken Chemicals, Tokyo, Japan) against 84 strains of Staphylococcus aureus, consisting of 82 strains of methicillin-resistant S. aureus (MRSA) from clinical isolated, S. aureus Mu3 involving beta-lactam antibiotic induced vancomycin (VCM) resistant MRSA (BIVR) and methicillin-susceptible S. aureus ATCC 29213. The results were in good accordance with the values determined by Clinical and Laboratory Standards Institute (CLSI): i.e., 100% (84/84) of consistency for VCM and 95% (80/84) for TEIC, respectively. In addition, BIVR strains were properly estimated from the results of the CA method and using the BIVR detection method with Mu3 agar (Mu3 Agar method), even though the incubation times was very short (2-4 h). In conclusion, it was found that the new method is reliable and rapid to detect BIVR strains in clinical laboratories.

During the period from January through December 2007, a total of 1,814 stool specimens from the inpatients of nine regional hospitals in mainland of the Ryukyus, were tested to identify vancomycin-resistant enterococci (VRE). All the stool specimens were primarily cultured onto the VRE selective agar plates, and a total of 195 specimens yielded positive enterococcal growth. Of 195 isolates, VRE screening agar tests identified 106 phenotypic VRE isolates, consisting 24 isolates of Enterococcus casseliflavus, 12 of E. faecalis, 4 of E. faecium, and 66 of E. gallinarum. Then, the 106 VRE isolates were tested for vanA and vanB genes by polymerase chain reaction, the results indicating none of the isolates were positive for vanA or vanB genes. With these results, it can be concluded that, at present, mainland of the Ryukyus is VRE-free area, and it is necessary to continue careful search for incoming and spreading of VRE positive for vanA and vanB genes in Okinawa.

Clostridium perfringens (C. perfringens) is a Gram-positive bacterial pathogen that widely propagets in the soil and the gastrointestinal tract of human and animals. This bacteria causes food poisoning, gas gangrene and other various range of infectious diseases. But there is no standard diagnosis method of C. perfringens. In order to develop a new type of immunoassay for clinical purpose, we studied expression and extracellular secretion of recombinant alpha-toxin having enzyme activity in E. coli expression system. Cloning was carried out after PCR amplification from C. perfringens GAI 94074 which was clinical isolate. Three kinds of fragment were cloned using pET100/D-TOPO vector. These fragments coded for ribosome binding site, signal peptide, and alpha-toxin gene respectively. Recombinant pET100 plasmid transformed into TOP 10 cells and the obtained plasmids were transformed into BL21 (DE3) cells. Then, the transformants were induced expression with IPTG. In conclusion, we successfully cloned, expressed and exteracellular secreted C. perfringens alpha-toxin containing signal peptide. Biologically, the obtained recombinant protein was positive for phospholipase C activity.

For the rapid and sensitive detection of influenza A and B viruses, a latex turbidimetric immunoassay (LTIA) was developed using latex reagents prepared by the sensitization of anti-influenza A or B monoclonal antibodies on latex particles. We measured the immunoreactivity of these latex reagents to influenza A and B viral antigens. The sensitivity and specificity of LTIA and reverse transcription-polymerase chain reaction (RT-PCR) for the detection of these viruses in clinical specimens (96 nasal swabs) were compared. The absorbance change in the latex agglutination reaction increased for each latex reagent with increasing concentration of the viral antigens. Reaction curves were obtained with each concentration of viral antigens for 5 min. The effective concentration ranges were 0-10 microg/ml for influenza A and 0-20 microg/ml for influenza B. The LTIA using clinical specimens revealed 8 positive and 73 negative results for influenza A and 15 positive and 52 negative results for influenza B. The sensitivities and specificities were 89% (8/9) and 84% (73/87), respectively, for influenza A and 100% (15/15) and 64% (52/81), respectively, for influenza B. The corresponding positive predictive values (PPV) were 36% (8/22) for influenza A and 34% (15/44) for influenza B. The negative predictive values (NPV) were approximately 99% (73/74) for influenza A and 100% (52/52) for influenza B. The LTIA is a rapid and sensitive method for detection of the influenza virus; It can be used for high throughput assay by automatic measurement and can potentially be used during influenza pandemics.

The TOX A/B QUIK CHEK "NISSUI" which detects both toxin A (TcdA) and toxin B (TcdB) of Clostridium difficile in stool specimens through immunochromatography was first approved to be released in Japan, and we evaluated its accuracy. In the evaluation, the TOX A/B QUIK CHEK "NISSUI" could correctly detect TcdA and TcdB in solution and in stool specimens spiked with culture broth of TcdA and/or TcdB-producing isolates of C. difficile. The minimum detectable concentrations for TcdA and TcdB were determined to be < or =0.32 ng/ml and < or =0.63 ng/ml, respectively. The TOX A/B QUIK CHEK "NISSUI" gave the consistent results with the colon-endoscopic diagnosis, that is, all the 10 stool specimens from the patients with pseudomembranous colitis were read as being positive, but negative for five patients without any C. difficile-associated disease (CDAD). Of 10 positive stool specimens, one was read as being negative by the commercially available test reagents that can detect only TcdA. In clinical evaluation, a total of 240 stool specimens were tested. Of these, the TOX A/B QUIK CHEK "NISSUI" gave 19 positive results, and TcdA and/or TcdB-producing strains of C. difficile were successfully isolated from all the positive stool specimens, except one. Whereas, of 221 negative stool specimens, 28 isolates of C. difficile were recovered and 11 isolates were identified as TcdA and/or TcdB-producing strains. With these results, it can be concluded that the TOX A/B QUIK CHEK "NISSUI" can correctly detect both TcdA and TcdB of C. difficile, and should be promptly applied to clinical microbiology laboratory to make a definite diagnosis of CDAD, particularly for the CDAD caused by the TcdA-negative but TcdB-positive mutant strains.

Anoxomat Mart II (Mart Microbiology BV, Lichtenvooorde, Netherlands, Central Scientific Commerce Inc., Tokyo, Japan) is an anaerobic jar apparatus which uses a vacuum pump in combination with catalyst as gas replacement procedure to remove all traces of oxygen. As we had a chance to use Anoxomat Mart II, we compared it with other two anaerobic culture methods; namely AnaeroPack anaero (Mitsubishi Gas Chemical Co., Tokyo, Japan) which employs anaerobic jar method, and Concept400 (RUSKINN TECHNOLOGY LTD, England; Central Scientific Commerce INc., Tokyo, Japan) which uses anaerobic chamber method. We used 10 different species of anaerobic bacteria obtained from ATCC. One strain each of 10 species was cultured and examined for measurement of the sensitibity of an anaerobic indicator, th number of bacteria after 48 hour culture, the diameter of colonies, and MIC value. As a result, the time to reach the anaerobic condition was around 30 minutes by the Mart II against around 60 minutes by the AnaeroPack anaero. There was no difference concerning the number of bacteria after 48 hour culture among three methods. But anaerobic bacteria cultured by Mart II tended to make bigger colonies compared to other two methods in the 5 strains out of 9, except for one strain in which the diameter of colonies could not be measured. On the other hand, the comparison of MIC value showed good correlation in 11 antibiotics out of 12 among three methods. The MIC value of 11 antibiotics fitted within 1-fold difference, and 2-fold difference was observed in only one antibiotic. Mart II is so small that it does cheep consumables. From these reasons, we concluded that Mart II can be one of the useful anerobic culture methods.