Assessing the discriminability of PCR-based open reading frame typing versus single-nucleotide polymorphism analysis via draft whole-genome sequencing of methicillin-resistant Staphylococcus aureus in nosocomial transmission analysis.

Phylogenetic analysis based on single-nucleotide polymorphism (SNP)-based through whole-genome sequencing is recognized as the standard method for probing nosocomial transmission. However, the application of WGS is constrained by the high cost of equipment and the need for diverse analysis tools, which limits its widespread use in clinical laboratory settings. In Japan, the prevalent use of PCR-based open reading frame typing (POT) for tracing methicillin-resistant Staphylococcus aureus (MRSA) transmission routes is attributed to its simplicity and ease of use. Although POT's discriminatory power is considered insufficient for nosocomial transmission analysis, conclusive data supporting this notion is lacking. This study assessed the discriminatory capabilities of SNP analysis and POT across 64 clinical MRSA strains. All 21 MRSA strains of ST5/SCCmec IIa, having more than 16 SNPs, demonstrated distinct clones. Conversely, two strains shared the same POT number and were identified as group A. Among the 12 MRSA strains of ST8/SCCmec IVl with over nine SNPs, five fell into POT group B, and five into POT group C. All four MRSA strains of ST8/SCCmec IVa were classified into POT group D, although they included strains with more than 30 SNPs. Among the 27 MRSA strains of ST1/SCCmec IVa, 14 were classified into POT group E. However, except for two clusters (each comprising two or three strains), all had SNP counts >10 (Fig. 1-D). SNP analysis of MRSA in CC1/SCCmec IV showed that several strains had the same number of SNPs in POT number (106-183-37), even among bacteria with >100 SNPs, indicating POT's limited use in detailed nosocomial transmission analysis.

Evolutionary dynamics of the novel ST22-PT methicillin-resistant Staphylococcus aureus clone co-harbouring Panton-Valentine leucocidin and duplicated toxic shock syndrome toxin 1 genes.

OBJECTIVES: Globally, the isolation of community-associated methicillin-resistant Staphylococcus aureus (MRSA) harbouring both the Panton-Valentine leucocidin (PVL) and toxic shock syndrome toxin 1 (TSST-1) genes is rare. However, we encountered an outbreak of the ST22-PT clone exhibiting this phenotype in Japan. Notably, the TSST-1 gene was duplicated in most of the strains. This study aimed to elucidate the mechanisms underlying this gene duplication. METHODS: A total of 90 MRSA isolates were collected from the skin of outpatients in Fukuoka City, Japan, between 2017 and 2019. Whole-genome sequencing was performed on MRSA strains that were PVL and TSST-1 positive. RESULTS: A total of 43 (47.8%) strains produced TSST-1, 20 (22.2%) produced PVL, and 16 (17.8%) produced both. Fifteen isolates were classified as ST22/SCCmec type IVa (ST22-PT clone) and one as ST1/SCCmec type V (ST1-PT clone). Three distinct ST22-PT clones were identified: Fukuoka clone I (one PVL gene and one TSST-1 gene), Fukuoka clone II (addition of a TSST-1 gene to Fukuoka clone I), and Fukuoka clone III (marked by a chromosomal inversion in a large region from Fukuoka clone II). DISCUSSION: Fukuoka clone I may have integrated a novel pathogenicity island bearing the TSST-1 gene, leading to the emergence of Fukuoka clone II with a duplicated TSST-1 gene. This duplication subsequently instigated a chromosomal inversion in a large region owing to the homologous sequence surrounding TSST-1, giving rise to Fukuoka clone III. These findings provide crucial insights into the genetic evolution of MRSA.

Whole-Genome Sequencing of the Novel Pseudomonas Strain TUM22785 Harboring blaPAM-1, Isolated from a Patient with Urinary Tract Infection in Japan.

Pseudomonas species are Gram-negative aerobic bacteria that cause opportunistic infections. Here, we report the whole-genome sequence of the Pseudomonas sp. strain TUM22785, isolated from an outpatient with a urinary tract infection at a medical institution in Japan. This strain harbors a metallo-ß-lactamase (MBL) blaPAM-1 gene.

Is the information on infection prevention measures against COVID-19 reaching the target audience? A cross-sectional survey among eating and drinking services in Tokyo, Japan.

Objectives: The study aims to evaluate information gathering behaviour (IGB) and its effectiveness in eating and drinking services for infection control during COVID-19. Study design: A cross-sectional survey using anonymous self-administered questionnaires was conducted in October 2021. Participants were asked what IGB they use to obtain infection control measures, to what extent they understand the measures (and, if they do not understand them, what inhibits their comprehension), and which IGBs they do not currently use and why. Methods: The sample included 957 eating and drinking services in Ota City, Tokyo. The response rate was 14.5%. Binomial logistic regression was used to analyse the factors associated with the baseline characteristics using Stata v.17.0. Results: The highest proportion of respondents used television (88.0%); another large proportion (38.9%) used guidelines. Regarding difficulty in understanding the retrieved information, 'difficulty in coming up with specific actions' had the highest ratio for every IGB. Regarding reasons for not currently using IGB, 'it takes too much time to extract the necessary information' showed the highest ratios of all IGBs. Individuals over 60 years had a negative relationship with the use of guidelines and the Internet. Participants also advised that they did not use time-consuming guidelines. Conclusion: Current information dissemination methods for information on COVID-19 infection control may not successfully convey information or reach their target populations. This study indicates the need for specific expressions and layouts to effectively share information on COVID-19. Also, special means of communication must be established to cater to individuals aged 60 and above.

Performance evaluation of Novaplex SARS-CoV-2 variants assay kit series for SARS-CoV-2 detection using single nucleotide polymorphisms.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have received increasing attention globally because of their increased transmissibility and potential to escape immunity. Although whole-genome sequencing is the gold standard method for SARS-CoV-2 mutation detection and lineage determination, it is costly and time-consuming. However, SARS-CoV-2 variants can be identified based on select variant-specific single nucleotide polymorphisms (SNPs) in the spike protein-encoding gene (S). This study validated and compared the limit of detection (LOD) of L452R, N501Y, HV69/70 del and E484K as variant-specific SNPs of the S gene and RdRP as a SARS-CoV-2-specific gene, using the Novaplex SARS-CoV-2 variants assay kit series. For three SARS-CoV-2 lineages (B.1.617.2, B.1.1.7 and R.1), one strain per lineage was used. Variant-specific SNPs of the S gene were analysed using the Novaplex SARS-CoV-2 variants I assay and Novaplex SARS-CoV-2 variants II assay kits. Validation confirmed the LODs of the variant kits. The LOD for each target variant-specific SNP and RdRP was five RNA copies per reaction. The Novaplex SARS-CoV-2 variants assay kit series performs well and the LOD for SARS-CoV-2 detection and variant-specific SNP detection are consistent. The kits are suitable for use as routine laboratory tests for SARS-CoV-2 and variant-specific SNP detection in a single step, saving time and labour.

Stereo sampling moiré method for three-dimensional deformation mapping with a stereomicroscope.

Three-dimensional (3D) deformation distribution measurement is of great interest in applications of materials evaluation. In this study, we propose a stereo sampling moiré method for full-field 3D deformation measurement based on a stereomicroscope. From the phase analysis on the left and right image planes as well as the relationship between displacements in the world and image coordinate systems, the 3D displacements and the in-plane strains of the specimen can be acquired. We also propose a method to calibrate the microscope convergence angle using the grid pitch variation. The validation experiment shows that the difference between the out-of-plane displacement measured by the proposed method and the movement of the sample stage is less than 0.2 µm. The microscopic 3D displacements and the in-plane strain distributions of a carbon fiber reinforced plastic specimen in a three-point bending test are investigated.

Multiplication sampling moire method for full-field deformation measurement of composite materials.

A multiplication sampling moire (MSM) method was proposed for robust deformation distribution measurement by performing phase analysis of the second harmonic (second-order frequency) of a single grating pattern. The MSM method has a very strong noise immunity because the second harmonic spectrum is far from the low-frequency region of the background noise in the frequency domain. Phase analysis of an experimental grid image on a carbon fiber-reinforced plastic (CFRP) specimen indicated that the MSM method effectively solved the problem of non-negligible phase measurement errors of conventional methods that extract the fundamental frequency of the grating, in the case of severe local noise. The displacement and strain distributions of CFRP in a tensile test were successfully measured. This method is suitable for deformation measurement of various composite materials.

Detection of SARS-CoV-2 RNA Using RT-qPCR in Saliva Samples and Nasopharyngeal, Lingual, and Buccal Mucosal Swabs.

Coronavirus disease 2019 is diagnosed based on the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in nasopharyngeal swabs or saliva samples using reverse-transcription quantitative polymerase chain reaction. Nasopharyngeal swabs should be collected by medical professionals who are covered with full personal protective equipment (PPE), while saliva samples can be collected by patients themselves without any PPE. However, collecting saliva is difficult for people who are unable to follow instructions, including infants or unconscious patients. Owing to the high viscosity of saliva, special attention is required to handle saliva samples in laboratories. To solve these problems, we compared lingual and buccal mucosal swabs (oral swabs) with nasopharyngeal swabs and saliva samples. Among 13 patients who had a positive result for SARS-CoV-2 RNA in their nasopharyngeal swabs, 8 and 10 patients had a positive result for SARS-CoV-2 RNA in their saliva (concordance rate, 61.5%) and oral swabs (76.9%), respectively. Among the eight patients with a positive result for SARS-CoV-2 RNA in saliva, seven (87.5%) had SARS-CoV-2 detected in their oral swabs. We could not obtain saliva samples from four patients, but we found perfect concordance of SARS-CoV-2 positivity between the nasopharyngeal and oral swabs. Therefore, oral swabs can be used for SARS-CoV-2 RNA detection.

Numerical and Experimental Studies for Fatigue Damage Accumulation of CFRP Cross-Ply Laminates Based on Entropy Failure Criterion.

The transverse cracking behavior of a carbon-fiber-reinforced plastic (CFRP) cross-ply laminate is investigated using a fatigue test and an entropy-based failure criterion in this study. The results of fatigue experiments show that the crack accumulation behavior depends on the cyclic number level and frequency, in which two obvious transverse cracks are observed after 104 cyclic loads and 37 transverse cracks occur after 105 cycles. The final numbers of transverse cracks decrease from 29 to 11 when the load frequency increases from 5 Hz to 10 Hz. An entropy-based failure criterion is proposed to predict the long-term lifetime of laminates under cyclic loadings. The transverse strength of 90° ply is approximated by the Weibull distribution for a realistic simulation. Progressive damage and transverse cracking behavior in CFRP ply can be reproduced due to entropy generation and strength degradation. The effects of stress level and load frequency on the transverse cracking behavior are investigated. It is discovered that, at the edge, the stress σ22 + σ33 that is a dominant factor for matrix tensile failure mode is greater than the interior at the first cycle load, and as stress levels rise, a transverse initial crack forms sooner. However, the initial transverse crack initiation is delayed as load frequencies increase. In addition, transverse crack density increases quickly after initial crack formation and then increases slowly with the number of load cycles. The proposed method's results agree well with those of the existing experimental method qualitatively. In addition, the proposed entropy-based failure criterion can account for the effect of load frequency on transverse crack growth rate, which cannot be addressed by the well-known Paris law.

Association between the point-rating system used for oral health and the prevalence of Gram-negative bacilli in hematological inpatients: A retrospective cohort study.

ABSTRACT: Gram-negative bacteremia is a major cause of death among hematology inpatients who require heavy-dose chemotherapy and hematopoietic stem cell transplantation. Gram-negative bacillus (GNB) is more likely to be detected when the oral health is poor. However, there is a dearth of studies on the relationship between oral assessment and prevalence of GNB in hematology inpatients.This retrospective study aimed to evaluate the relationship between the original point-rating system for oral health examinations (point-oral exam) and the prevalence of GNB in hematology inpatients at the hematology ward of the Yamanashi University Hospital. GNB was detected by cultivating samples from the sputum and blood of each patient.A total of 129 subjects underwent a medical checkup and point-oral exam. The sputum and blood culture results of 55 patients were included in this study. The total points of patients positive for GNB (n = 25, 45.5%) were significantly higher than those who were negative for GNB (total score: median, 25th, 75th, percentile; 6 [4, 7] vs 2 [1, 4]; P = .00016). Based on the receiver operating characteristic analysis, a cutoff score of 5 proved to be most useful to detect GNB.An oral evaluation with a cutoff value of 5 or higher in the point-oral exam might indicate the need for a more thorough oral management to prevent the development of systemic infections from GNB.

Apparent Life-Threatening Event in an Infant with SARS-CoV-2 Infection.

The 2019 novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused a global outbreak of infection. In general, children with coronavirus disease-2019 have been reported to show milder respiratory symptoms than adult patients. Here, we have described a case of a SARS-CoV-2-infected infant who presented to our hospital with a severe episode of an apparent life-threatening event (ALTE). An 8-month-old, otherwise healthy female infant presented to our hospital because of a sudden cardiopulmonary arrest. Approximately 1 h before this episode, the patient showed no symptoms, except a worse humor than usual. On arrival at our hospital, the patient had severe acidosis, but there were no clear signs of inflammatory response. Chest computed tomography showed weak consolidations in the upper right lung and atelectasis in the lower left lung. No signs of congenital heart disease or cardiomyopathy were observed on echocardiography, and no significant arrhythmia was observed during the clinical course. However, SARS-CoV-2 RNA was detected by real-time reverse transcription polymerase chain reaction in tracheal aspirate and urine samples. Although the assessment of further similar cases is indispensable, this case suggests that SARS-CoV-2 infection may be an underlying factor in the pathophysiology of ALTE.

Clinical characteristics and molecular analysis of USA300 and ST 764 methicillin-resistant Staphylococcus aureus isolates from outpatients in Japan by PCR-Based open reading frame typing.

INTRODUCTION: USA300 is the most common community-acquired methicillin-resistant Staphylococcus aureus (MRSA) strain. Sequence type (ST) 764 MRSA is a new local variant of the ST 5 lineage. The objective of this study was to determine the clinical characteristics of USA300 and ST 764 infections among outpatients in Japan. METHODS: We obtained MRSA isolates from 132 outpatients who visited our hospital from January 2016 to December 2017 and compared USA300 infection group to ST 764 infection group. Molecular analysis, including that of various toxins and other virulence factors, of the MRSA isolates were performed. In particular, we investigated the relationships among PCR-based open reading frame typing (POT) scores, MRSA clones, and virulence factors. RESULTS: Twenty-seven USA300 isolates (20.5%) and 16 ST 764 isolates (12.1%) were identified. Although USA300 and ST 764 had lower rates of risk factors, their infection rates were higher. USA300-infected patients had higher rates of deep skin and soft tissue infections compared with the non-USA300 CA-MRSA-infected patients. Notably, the USA300 and ST 764 isolates had unique POT scores. CONCLUSIONS: Our results indicated that USA300 MRSA was spreading in an area 120 km west of Tokyo, Japan. We observed multiple cases of ST 764 MRSA infection, raising concerns about the antimicrobial resistance of ST 764, as it limits the choices of antibiotics to treat infection. The POT score can predict the presence of toxins and virulence factors, as well as the clone identity of MRSA with high accuracy.

Second-order moiré method for accurate deformation measurement with a large field of view.

In this study, we propose a second-order moiré method by performing digital sampling at two stages to realize high-accuracy deformation measurement in a wide field of view, where a grid image is recorded at a low magnification. Simulations have verified that this method has high strain measurement accuracy when the grid pitch is close to or even smaller than two pixels for both parallel and oblique grids with random noise. As an application, the two-dimensional microscale strain distributions of a carbon fiber reinforced plastic specimen when the grid pitch was about 2.1 pixels were presented. Shear strain concentration was detected before an interlaminar crack emerged, and tensile strain concentration was found prior to the occurrence of a transverse crack. The proposed method makes the two-step phase-shifting technique achieved indirectly, not only enlarging the field of view, but also maintaining the measurement accuracy.

A first case of meningitis/encephalitis associated with SARS-Coronavirus-2.

Novel coronavirus (SARS-Coronavirus-2:SARS-CoV-2) which emerged in Wuhan, China, has spread to multiple countries rapidly. We report the first case of meningitis associated with SARS-CoV-2 who was brought in by ambulance due to a convulsion accompanied by unconsciousness. He had never been to any foreign countries. He felt generalized fatigue and fever (day 1). He saw doctors nearby twice (day 2 and 5) and was prescribed Laninamivir and antipyretic agents, His family visited his home and found that he was unconsciousness and lying on the floor in his vomit. He was immediately transported to this hospital by ambulance (day 9). Under emergency transport, he had transient generalized seizures that lasted about a minute. He had obvious neck stiffness. The specific SARS-CoV-2 RNA was not detected in the nasopharyngeal swab but was detected in a CSF. Anti- HSV 1 and varicella-zoster IgM antibodies were not detected in serum samples. A brain MRI showed hyperintensity along the wall of right lateral ventricle and hyperintense signal changes in the right mesial temporal lobe and hippocampus, suggesting the possibility of SARS-CoV-2 meningitis. This case warns the physicians of patients who have CNS symptoms.

Interlaminar Shear Behavior of Laminated Carbon Fiber Reinforced Plastic from Microscale Strain Distributions Measured by Sampling Moiré Technique.

In this article, the interlaminar shear behavior of a [±45°]4s laminated carbon fiber reinforced plastic (CFRP) specimen is investigated, by utilizing microscale strain mapping in a wide field of view. A three-point bending device is developed under a laser scanning microscope, and the full-field strain distributions, including normal, shear and principal strains on the cross section of CFRP, in a three-point bending test, are measured using a developed sampling Moiré technique. The microscale shear strain concentrations at interfaces between each two adjacent layers were successfully detected and found to be positive-negative alternately distributed before damage occurrence. The 45° layers slipped to the right relative to the -45° layers, visualized from the revised Moiré phases, and shear strain distributions of the angle-ply CFRP under different loads. The absolute values of the shear strain at interfaces gradually rose with the increase of the bending load, and the sudden decrease of the shear strain peak value implied the occurrence of interlaminar damage. The evolution of the shear strain concentrations is useful in the quantitative evaluation of the potential interlaminar shear failure.

Molecular typing of methicillin-resistant Staphylococcus aureus: Comparison of PCR-based open reading frame typing, multilocus sequence typing, and Staphylococcus protein A gene typing.

The recently developed PCR-based open reading frame typing (POT) method is a useful molecular typing tool. Here, we evaluated the performance of POT for molecular typing of methicillin-resistant Staphylococcus aureus (MRSA) isolates and compared its performance to those of multilocus sequence typing (MLST) and Staphylococcus protein A gene typing (spa typing). Thirty-seven MRSA isolates were collected between July 2012 and May 2015. MLST, spa typing, and POT were performed, and their discriminatory powers were evaluated using Simpson's index analysis. The MRSA isolates were classified into 11, 18, and 33 types by MLST, spa typing, and POT, respectively. The predominant strains identified by MLST, spa typing, and POT were ST8 and ST764, t002, and 93-191-127, respectively. The discriminatory power of MLST, spa typing, and POT was 0.853, 0.875, and 0.992, respectively, indicating that POT had the highest discriminatory power. Moreover, the results of MLST and spa were available after 2 days, whereas that of POT was available in 5 h. Furthermore, POT is rapid and easy to perform and interpret. Therefore, POT is a superior molecular typing tool for monitoring nosocomial transmission of MRSA.

First Japanese case of infectious endocarditis due to Enterococcus faecalis small-colony variants.

A male patient was admitted to our hospital due to infectious endocarditis. He had been treated with levofloxacin for 6 weeks, sulbactam/cefoperazone for 4 weeks, and benzylpenicillin for 2 days prior to valve replacement surgery. Gram-positive cocci, with morphology consistent with γ-Streptococcus, were detected in blood cultures obtained at admission, as well as in vegetation obtained from the aortic valve. However, the strain could not be identified using biochemical methods. Sequencing of the 16S rRNA gene indicated that the culture was a small-colony variant of Enterococcus faecalis. This is the first case in Japan of infectious endocarditis due to E. faecalis small-colony variants. Small-colony variants are subpopulations of bacteria with slow growth, reduced sugar fermentation, and unstable phenotype. As a result, these strains tend to be misidentified. Further, small-colony variants are associated with recurrent and persistent infections such as prosthetic joint infection and infectious endocarditis. These strains are found in various bacterial species, including Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa, but rarely in Enterococcus species. The case highlights the need to be vigilant of E. faecalis small-colony variants, especially in patients who received long-term courses of antibiotics.

Rapid assay of A2058T-mutated 23S rRNA allelic profiles associated with high-level macrolide resistance in Moraxella catarrhalis.

We report on a restriction fragment-length polymorphism (HpyCH4III) assay for profile analysis of 23S rRNA gene A2058T-mutated alleles associated with high-level macrolide resistance in Moraxella catarrhalis. Our assay results were supported by DNA sequencing analysis, allowed for simultaneous testing of many strains, and produced results from pure-cultured colonies within 4 h.

Characterization of Mucoid and Non-Mucoid Streptococcus pneumoniae Isolated From Outpatients.

BACKGROUND: Streptococcus pneumoniae causes pneumonia, sepsis, and meningitis. This study aimed to investigate the clinical characteristics of mucoid and non-mucoid isolates of S. pneumoniae, and to explore the relationship between the isolate phenotypes and their antibiotic susceptibility. METHODS: Clinical isolates from 3,453 non-repetitive S. pneumoniae (189 mucoid and 3,264 non-mucoid) infections obtained between January 2008 and December 2012 from outpatients at the Kimitsu-Central Hospital were evaluated. RESULTS: Compared to the non-mucoid isolates, the mucoid phenotypes were more susceptible to certain antibiotics such as erythromycin, clarithromycin, and tetracycline as opposed to clindamycin, chloramphenicol, and rifampicin. The mucoid phenotype was isolated more frequently from schoolchildren, adults, and elderly adults in a variety of clinical sites, including otorrhea, genitalia, pus, and eye discharge than the non-mucoid phenotype. This suggested that mucoid isolates are more likely to be involved than non-mucoid isolates in various local infections. Systemic infection, which indicates invasiveness, was not associated with the mucoid or non-mucoid phenotype. CONCLUSIONS: The results of this study suggest that mucoid isolates tend to have higher susceptibility than non-mucoid isolates to antibiotics. To the best of our knowledge, mucoid and non-mucoid S. pneumoniae isolates considerably differ in terms of clinical isolation site and age-specific prevalence.

Prevalence and molecular analysis of macrolide-resistant Moraxella catarrhalis clinical isolates in Japan, following emergence of the highly macrolide-resistant strain NSH1 in 2011.

Although Moraxella catarrhalis is known to be susceptible to macrolides, highly macrolide-resistant M. catarrhalis isolates have recently been reported in Japan and China. In this study, we investigated the prevalence of macrolide-resistant M. catarrhalis isolates in Tokyo and Chiba, Japan, and studied the mechanisms underlying their resistance. Specifically, we determined the susceptibility of 593 clinical isolates (collected between December 2011 and May 2014) to erythromycin, using the disk diffusion method. For isolates with erythromycin resistance, we identified the MICs of seven antimicrobial agents, including macrolides, and used PFGE to analyse the clonal spread. We also performed sequencing analysis to investigate macrolide-resistance targets. Thirteen isolates (2.2 %) were found to be resistant to erythromycin, showing a high MIC90 to erythromycin, clarithromycin, clindamycin and azithromycin. However, those isolates, in addition to 156 randomly selected erythromycin-susceptible strains, were susceptible to amoxicillin-clavulanate, cefixime and levofloxacin. The 13 highly macrolide-resistant isolates were classified into 10 clades and harboured three or four A2058T-mutated 23S rRNA alleles. Three highly macrolide-resistant isolates also exhibited mutations in ribosomal proteins L4 (V27A and R161C) and L22 (K68T). To the best of our knowledge, we have demonstrated for the first time that, whilst the prevalence of macrolide-resistant M. catarrhalis isolates is low in clinical settings in Japan, genetically diverse isolates with high-level macrolide resistance due to the acquisition of an A2058T mutation in the 23S rRNA have already spread. Our study therefore lays the basis for epidemiological studies of macrolide-resistant M. catarrhalis clinical isolates.