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

For diagnosis of Mycoplasma pneumoniae infection, highly sensitive and rapid diagnosis is important. Because antibiotics are limited for the treatment of M. pneumoniae infection. In this study, we evaluated new rapid nucleic acid detection kit for M. pneumoniae. This kit does not require excessive pretreatment of specimens and molecular diagnosis of M. pneumoniae is possible within 40 min. Using 120 nasopharyngeal specimens, we compared this kit with a commercially available molecular diagnostic reagent (LAMP). 51 of 120 cases were M. pneumoniae positive, and the results of both assays were all consistent. In addition, sequencing of 23S rRNA gene was performed on 51 cases positive for M. pneumoniae. As a result, macrolide resistance mutation (2063A>G) was observed in 19 cases (37.3%). The gene mutations estimated by this kit coincided completely with the sequencing. In conclusion, new rapid nucleic acid detection kit could detect M. pneumoniae with the same sensitivity as other molecular diagnostics, in a simple process.

We evaluated performance of Versa TREK, blood culture system used in our hospital. Compared with BacT/ALERT 3D, the detection time of bacteria in the VersaTREK was shorter in most of strains. Compared with BacT/ALERT Virtuo, there was little difference in the detection time of bacteria. In addition, VersaTREK was able to detect Helicobacter cinaedi which could not be detected by other equipment, and H. cinaedi was detected in clinical specimens within 2 days. There were 147 bottles judged to be false positives at our facility, of which 7,290 were 2,0% of the total. Ninety one points eight percentage of the cause was due to the change in the temperature inside the device, 3.4% was due to incorrect procedure. So, it is considered that false positives are further decreased by appropriate management of the installation and sample collection.

In bacterial identification by MALDI-TOF MS, there are many reports of usefulness concerning direct identification from blood culture and identification of bacteria which cannot be identified with automatic analysis equipment. On the other hand, there are very few studies that investigate how various conditions influence on identification accuracy, such as the type of medium used for bacterial isolation and pure culture, the pretreatment methods, the difference in coating technique, and preservation methods. Therefore, we examined 10 strains of 2 drug-resistant bacteria species and 9 strains of 1 unnormal bacterium species. As a result, no significant differences were found in accuracy of identifying all strains of the target bacteria incubated for 24 hours and changing the types of medium, the pretreatment methods, and the coating techniques. In particular, methicillin-resistant Staphylococcus aureus (MRSA) and extended-spectrum β-lactamase (ESBL) producing Escherichia coli showed little change in the score value and the mass spectrum that assayed every 24 hours during the preservation period in all of the medium. In the case of Vibrio vulnificus, however, identification accuracy was decreased by the specific medium and storage conditions. It is suggested as this factor that the growth state of bacteria may have influenced the identification accuracy.

As the reagent that can simultaneously detect bacterial nucleic acid/drug-resistant genes from the culture-positive liquid of blood cultures, Verigene^® system includes the Verigene^® Gram-Positive Blood Culture test (BC-GP) and the Verigene^® Gram Negative Blood Culture test (BC-GN). This study used BC-GN to identify the names of bacteria from stock strains, urine samples, and sputum specimens and detect drug-resistant genes. The stock strains included 28 clinical isolates, 9 urine samples in which the target bacterial strain grew to 106CFU/ml or more in culture, and 9 sputum specimens in which the target bacterial strain grew to 105CFU/ml or more in culture. The bacterial identification and detection of drug-resistant genes used quality Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) analysis and conventional PCR method, respectively, followed in comparison with the results of Verigene^®. As a result, the measurement results of Verigene^® for the stock strains and urine samples had a high concordance rate with MALDI-TOF MS analysis and PCR method. On the other hand, the concordance rate of the sputum specimens with the Verigene^® measurement results was only 40% (4 out of 10 specimens). These results suggest that BC‒GN can be an effective tool for AMR rapid diagnosis if the measurement target includes not only bacterial strains in the culture-positive liquid of blood cultures, but also other bacterial strains and urine.

Reliable detection and typing of carbapenemase is important in the treatment of infectious diseases. In this study we newly designed LAMP primer based on the latest information, and established a detection method for Carbapenemase Big five gene. For DNA extraction from strains, alkaline boiling method and commercial kit were used. The reaction temperatures of the LAMP method was VIM: 65°C, NDM: 63°C, KPC: 65°C, OXA-48-like: 65°C, IMP: 61°C. And simultaneous LAMP method was at 63°C, for 60 min. It was possible to detect up to 10³ copies/ml. The reactivity of LAMP using 36 strains verified by Multiplex-PCR was VIM (4/4: number of LAMP method positive strains/number of strains evaluated), NDM (2/2), KPC (4/4), OXA-48-like (4/4), IMP (17/17). The type of carbapenemase determined by the LAMP method were all consistent with multiplex PCR. All strains were detected within 30 min. In VIM, both VIM-1-like and VIM-2-like were able to detect. In this study, although the number and variation of the strains evaluated was limited, LAMP method was clinically useful as a simple and rapid carbapenemase detection method.

In order to develop a new method to detect β-lactamase, we determined degradation of β-lactam antibiotics, ampicillin sodium (ABPC) and cefotaxime sodium (CTX), by β-lactamase-producing Klebsiella pneumoniae (K. pneumoniae) in terms of high performance liquid chromatography (HPLC). Using HPLC with an ODS column and an eluent composed of phosphate buffer and methanol, we could detect ABPC and CTX within 10 min. After cultured with K. pneumoniae, ABPC and CTX were degraded. The degradation rate corresponding to the rate of peak area incubated with and without bacteria increased with increasing McFarland No. of bacterial suspension and incubation time. Under the culture condition of McFarland No. 3.0 and 90-min incubation, the degradation rate of ABPC and CTX was 52.6 and 70.8% by K. pneumoniae, whereas it was less than 10% by susceptible bacteria. Consequently, these results confirmed that β-lactamase produced by ABPC- and CTX-resistant bacteria could be detected within about 120 min through HPLC measurement.

Legionella pneumophila, the causative agent of Legionnaires' disease, was first recognized in 1977 following an epidemic of acute pneumonia in Philadelphia, USA. Since then, a total of 59 Legionella species containing 80 serogroups have been characterized. Twenty-six of the Legionella species have been reported as pathogenic in humans. This review describes the microbiological characteristics of Legionella species, their habits in the environment, the source and route of infection, symptoms and diagnosis of Legionnaires' disease, and disease outbreaks in Japan.

In recent years, the appearance of MALDI-TOF MS made it possible to identify bacterial species in a short time. However, the sensitivity test has not largely shortened the time. When ESBL is suspected by routine examination, we speculate that there are many institutions conducting the inspection method conforming to the CLSI inspection procedure. When carrying out bacteria identification, susceptibility test and ESBL confirmation test from blood culture-positive specimens, the expected examination days are required for about 3 to 4 days, which is not quick. We report on bacterial species identification by MALDI-TOF MS from blood culture positive specimen bottle and ESBL rapid decision with fully automated urine analyzer.

Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira spp. The severity of leptospirosis vary from mild, flu-like disease to a more severe form, Weil's disease causing jaundice, hemorrhage, renal failure, and even death. Every year, 300,000‒500,000 cases of severe leptospirosis are reported around the world, with the case fatality rate being 10‒30%. The usual diagnostic tools for leptospirosis are 1) direct observation of leptospires in blood and urine under dark-field microscope, 2) isolation of leptospires from blood, cerebrospinal fluid (CSF), or urine samples by culture, 3) microscopic agglutination test (MAT) to detect anti-Leptospira antibodies in serum, and 4) PCR to detect Leptospira DNA. At presents, the gold standards for diagnosis are culture isolation and MAT. However, it is actually not easy to isolate leptospires from clinical samples. On the other hand, it takes several days before the results of MAT become positive after the onset of illness. Moreover, MAT requires skilled handling, and also needs the maintenance of live Leptospira cells representing all serogroups. Hence other simple or rapid diagnostic tests are needed at the bedside. The micro capsule agglutination test (MCAT) to detect antibody and immunochromatographic assay to detect urinary antigen are currently in the research and development phases. In this paper, the characteristics of each diagnostic test for leptospirosis are described.

Clinical and Laboratory Standards Institute (CLSI) methods for susceptibility tests of yeast are used in Japan. On the other hand, the methods have some disadvantage; 1) reading at 24 and 48 h, 2) using unclear scale, approximately 50% inhibition, to determine MICs, 3) calculating trailing growth and paradoxical effects. These makes it difficult to test the susuceptibility for yeasts. Old software of RAISUS, Ver. 6.0 series, resolved problem 1) and 2) but did not resolve problem 3). Recently, new software of RAISUS, Ver. 7.0 series, resolved problem 3). We confirmed that using the new software made it clear whether all these issue were settled or not. Eighty-four Candida isolated from Aichi Medical University was used in this study. We compared the MICs obtained by using RAISUS antifungal susceptibility testing of yeasts RSMY1, RSMY1, with those obtained by using ASTY. The concordance rates (±four-fold of MICs) between the MICs obtained by using ASTY and RSMY1 with the new software were more than 90%, except for miconazole (MCZ). The rate of MCZ was low, but MICs obtained by using CLSI methods and Yeast-like Fungus DP 'EIKEN' methods, E-DP, showed equivalent MICs of RSMY1 using the new software. The frequency of skip effects on RSMY1 using the new software markedly decreased relative to RSMY1 using the old software. In case of showing trailing growth, the new software of RAISUS made it possible to choice the correct MICs and to put up the sign of trailing growth on the result screen. New software of RAISUS enhances its usability and the accuracy of MICs. Using automatic instrument to determine MICs is useful to obtain objective results easily.