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

Clostridioides difficile is the most common anaerobic bacterium that causes healthcare-associated infections, and prompt diagnosis and infection control are important because it causes C. difficile infection (CDI). In this evaluation, the C. difficile nucleic acid detection reagent, Smart Gene CD Toxin B (Mizuho Medy Co., Ltd., hereinafter referred to as the "evaluation reagent") was evaluated for its clinical performance in comparison with real-time PCR and toxigenic culture (TC). Measurement of evaluation reagents and real-time PCR were performed on 157 residual stool specimens from suspected CDI patients. For TC, stool culture was performed, and colonies in which C. difficile was identified by a mass spectrometer (MALDI Biotyper) were checked for toxin production using a rapid antigen diagnostic kit. The results of the evaluation reagents showed a high concordance rate; 100% sensitivity (81/81) and 100% specificity (76/76) with real-time PCR, 89.8% sensitivity (79/88), and 97.1% specificity (67/69) with TC. The evaluation reagent enables a simple nucleic acid amplification test (NAAT) in a short time and is thought to be useful in CDI treatment, which requires rapid diagnosis and infection control.

Frequent mutations of SARS-CoV-2 change the strain more transmissible, leading to the pandemic in worldwide. We detected Y453F substitution on Omicron strain, isolated from a Japanese patient in July 2022. While Y453F substitution was identified B1.1.298 lineage in Netherlands and Denmark in 2020, the substitution has not been reported in Omicron strain especially in Japan. Y453F substitution is associated with higher viral infectivity because it is sited in the receptor-binding domain (RBD), and Y453F substitution contributes to increase binding affinity to angiotensin converting enzyme 2 (ACE2). Additionally, Y453F substitution has been reported to escape human leukocyte antigen (HLA), which is known to recognize non-self-antigens in virus-infected cells as cellular immunity, so it should be closely monitored.

Yersinia enterocolitica is a causative agent of food poisoning and has been isolated from pork and stream water, causing Yersinia enterocolitica in humans. The bacterium is divided into multiple serotypes and biotypes, among which serotypes O3 and O8 and biotypes 1B, 3, and 4 are frequently isolated in Japan. Biotype 3 can be classified as [VP+, Suc+], [VP-, Suc+], [VP-, Suc-] based on the biochemical properties. Among them, [O3, 3, VP-, Suc-] has been reported to be identified as Yersinia kristensenii in a simple identification kit. An increasing number of facilities in the field of microbiological testing are currently using mass spectrometers to identify species of microorganisms. However, there are many facilities where mass spectrometers have not yet been installed and microbial identification and susceptibility testing devices are used to identify bacterial species. No reports have described how the [O3, 3, VP-, Suc-] type, which is identified as Y. kristensenii in the simple identification kit, is identified by the microbial identification and susceptibility testing devices. In this study, 15 strains of Y. enterocolitica, which were previously isolated, serotyped, and biotyped from fecal culture tests at our hospital, were analyzed to see how these strains were identified in RAISUS S4, Microscan WalkAway, VITEK2 Blue, and BD Phoenix. [O3, 3, VP-, Suc-] was identified as Y. kristensenii in RAISUS S4, Microscan WalkAway, and VITEK2 Blue and as Y. enterocolitica in BD Phoenix. [O3, 3, VP-, Suc+], [O3, 4] and [O8, 1B] were identified as Y. enterocolitica. Therefore, when a sample was identified as Y. kristensenii by RAISUS S4, Microscan WalkAway, or VITEK2 Blue, the possibility that it was actually [O3, 3, VP-, Suc-] could not be ruled out. The possibility of Y. enterocolitica should be informed to attending physicians.

In this study, we examined the accuracy and rapidity of drug susceptibility determination using clinical isolates of MRSA with the fully automated rapid identification susceptibility testing system RAISUS S4. Ninety eight MRSA strains were used and the time until methicillin resistance was determined was analyzed by both of the standard method (18-hr method) and the rapid method. Five strains (5.1%) were determined to be methicillin-sensitive in MPIPC by rapid method only while all strains were determined to be resistant in CFX. The average methicillin resistance determination time was 7.0/5.0 hr for MPIPC, 6.3/5.0 hr for CFX, and 6.3/5.0 hr for the combination of MPIPC and CFX by the standard/rapid method, with the rapid method being significantly shorter (Wilcoxon's signed rank sum test, p<0.01). Strains determined to be methicillin-sensitive by MPIPC tended to have a longer time to methicillin resistance by the standard method, but this effect was much less pronounced for the rapid method using CFX. Methicillin resistance determination by the rapid method using RAISUS S4 enables rapid detection of MRSA without false-susceptible results, which may lead to early and appropriate treatment.

Methicillin-resistant Staphylococcus aureus (MRSA) strains showing POT type 106-77-113 have been associated with USA300. Additionally, many strains produce Panton-Valentine Leukocidin (PVL). Until 2018, 106-77-113 was the most dominant POT-type PVL-producing bacteria isolated in our hospital; however, in 2018, one strain with POT type 106-255-121 was isolated, and thereafter, since 2019, an increasing trend towards isolation of this strain has been observed. In this study, we compared two PVL-producing strains detected in skin infections-derived materials from outpatients during the three-year period between 2019 and 2021 through genetic analysis using next-generation sequencers. Eight, each of POT types 106-77-113 (POT-A) and 106-255-121 (POT-B), strains were included in this study, and PVL productivity, drug susceptibility, multi-locus sequencing typing (MLST), and resistance genes and virulence genes were detected. Both the groups shared the same MLST profile (3-3-1-1-4-4-3), but a single nucleotide mutation of ARCC was detected in POT-B type strain, which was determined to be similar to ST 8 of POT-A and POT-B type strains. Only POT-B type strains harbored aac(6')-aph(2″) and erm(A) genes, consistent with the results of drug susceptibility tests. All the strains were resistant to GM and CAM and were positive to D-zone test. On the other hand, the POT-A strains were sensitive to GM, and 7 of 8 strains were sensitive to CLDM and MINO. However, one POT-A type strain was found to harbor erm(C), tet(K), and tet(M) genes and was resistant to CAM, CLDM, and MINO. Both groups of isolates harbored 17 genes including ACME, lukF-PV, and LukS-PV, and no difference in pathogenicity was observed. In our hospital, one strain of POT type 106-255-121 was isolated for the first time in 2018, and the number of isolates of this type has been increasing since then. The present study confirms that POT type 106-255-121 strains have the same virulence as POT type 106-77-113 strains have and have also acquired a drug resistance gene.

We compared rapid antigen detection kits widely used for the rapid diagnosis of group A streptococcal pharyngitis, evaluating their minimum detection sensitivity and operability in five levels. Five kits based on the immunochromatographic method were used: ImunoAce Strep A (Tauns), ImunoAce Strep A Neo (Tauns), Quick Navi-StrepA2 (Denka), Quick Vue Dipstick Strep A (SB Bioscience) and RapidTesta Strep A (SEKISUI MEDICAL). Thirteen strains were tested: 10 clinical isolates of Streptococcus pyogenes, 2 strains of Streptococcus dysgalactiae subsp. equisimilis (SDSE), and S. pyogenes ATCC 19615. All kits had the same or higher minimum detection sensitivity than previously reported. ImunoAce StrepA Neo had the highest detection sensitivity and the best overall evaluation among the group A streptococcal rapid antigen detection kits used in this study. The detection sensitivity of SDSE with group A polysaccharide antigen was comparable to that of S. pyogenes. Although culture tests are necessary to confirm the causative organism, SDSE may present with clinical symptoms similar to those of S. pyogenes, and detection with a rapid antigen detection kit may be of therapeutic value.

Rapid positive blood culture reporting allows early and appropriate treatment of severe infections to improve patient prognosis. This study evaluated performance of the VersaTREK system with gas pressure detection and tornado stirring method and the conventional BacT/ALERT 3D system. Time to positivity (TTP) of simulated blood cultures without whole blood using 17 ATCC strains was faster with VersaTREK than BacT/ALERT 3D, averaging 6.3 h in aerobic bottles and 12.7 hours in anaerobic bottles. In simulated blood cultures with whole blood using 53 clinical isolates, on average, VersaTREK was faster in aerobic bottles by 6.5 h but slower in anaerobic bottles by 3.8 h. Fifty of 53 simulated blood cultures with whole blood (94%) showed fastest TTP with VersaTREK. TTP of VersaTREK for anaerobic bacteria Bacteroides fragilis and Clostridium perfringens, Helicobacter cinaedi, and Candida glabrata was fast, and viable bacteria numbers in bottles using the Miles and Misra method increased quickly.

Recently, the global spread of carbapenemase-producing Enterobacterales has become a concern, and rapid detection methods are required. We have developed a rapid and inexpensive multiplex real-time PCR with melting curve analysis using the BD MAX™ system and evaluated it. We used 31 carbapenemase-producing Gram-negative bacteria (bla_IMP group, 12; bla_GES group, 6; bla_NDM group, 5; bla_VIM group, 3; bla_KPC group, 3; bla_OXA-48-like group, 1 strain; and bla_IMP group + bla_GES, 1 strain), 10 AmpC-producing Gram-negative bacteria, and 10 ESBLproducing Gram-negative bacteria. A BD MAX™ open platform system was used. Carbapenemase-positive and carbapenemase-negative strains were correctly identified 30 of 31 (excluding a bla_IMP group and bla_GES group co-coding strain) and all 20 of 20 isolates, respectively. Melting temperature (Tm) values of the various genes were as follows: bla_IMP group, 81.2±0.5°C; bla_VIM group, 91.8±0.4°C; bla_NDM group, 85.4±0.3°C; bla_GES group, 90.5±0.3°C; bla_KPC group, 94.1±0.5°C; and bla_OXA-48-like group, 82.1°C. Identification of the various genotypes was possible from the Tm values. However, only a peak derived from the bla_GES group could be detected in the strains producing both bla_IMP group and bla_GES group simultaneously, suggesting that only the genotype with the highest expression level could be captured in cases of simultaneous production. In the carbapenemase-negative strains, no obvious peaks were observed in the 20 AmpC and 20 ESBL-producing Gram-negative bacteria, and even when Tm values were detected, the dF/dT values were low and easily differentiated. This method appears to be very useful as a rapid and inexpensive test that can provide detection in about 2 hours.

Introduction: From 2018, IR Biotyper (IRBT; Bruker Daltonik GmbH, Germany) based on the Fourier transform infrared spectrophotometer has begun to be introduced as a new strain classification method in the field of clinical microbiological examination. We compared it with molecular epidemiology method to evaluate the usefulness of strain classification by IRBT.

Method: Homology of strain classification with molecular epidemiology method (Multilocus Sequencing Typing; MLST and PCR-based ORF Typing; POT) for 48 strains of Pseudomonas aeruginosa with different detection times from multiple institutions to evaluate the accuracy of IRBT was compared.

Results: IRBT used "KBM" SCD agar medium for preculture and was classified into 12 types when classified by Cut-off value 0.181, 8 types by MLST, and 13 types by POT. In the Adjusted Wallace between IRBT and molecular epidemiology method, MLST was 0.458 (95% CI; 0.295 to 0.620) and POT was 0.330 (95% CI; 0.135 to 0.525), indicating a discrepancy in strain classification.

Conclusion: No correlation was found between IRBT and the classification results by the molecular epidemiology method. In the molecular epidemiology method, strains are classified by matching only specific gene regions, but IRBT irradiates a sample with an infrared laser and classifies the strains according to the difference in absorption spectrum according to the molecular structure, so the measurement principle is different. When classifying strains by IRBT, it is desirable to grasp the clinical information of the detected strains and to target multiple strains isolated at the same facility at the same time.

Rapid detection of carbapenemase-producing Enterobacterales (CPE) is important in infection control, since it transmits plasmids carrying resistance genes. Here, we evaluated the rapid detection of CPE using the fully automated antimicrobial susceptability testing system "RAISUS S4". Sixty-two CPE strains including carbapenem-resistant Enterobacterales and 100 carbapenemase-non-producing Enterobacterales strains were used. RAISUS S4 was performed using both 18 hr and rapid methods. The sensitivity of CPE detection and decision time were evaluated using Meropenem results. The results showed that the sensitivity for CPE detection was 100% for both methods, with specificity of 97% for the 18 hr method and 95% for the rapid method. The mean CPE detection time for the 18 hr method was 7.2 hrs and 8.8 hrs for the rapid method. The mean MIC determination time of the 18 hr method for all 162 strains was 17.2 hrs and 8.8 hrs for the rapid method. In addition, we analyzed the absorbance values of the 18 hr method. Checking the growth curve at a drug concentration of 0.125 µg/ml and determining it to be positive when its absorbance reached 0.8 Abs, CPE could be detected in an average of 5.8 hrs with in sensitivity of 100% and specificity of 92%. The 18 hr method of RAISUS S4 allowed rapid detection of CPE, and the rapid method allowed earlier MIC determination, including sensitive isolates. These results suggest that RAISUS S4 can detect CPE rapidly without missing CPE by routine test even in Japan where the frequency of CPE isolation is low.