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A 67-year-old woman with a 2-day history of a fever, headache and disturbed consciousness was admitted to our hospital. Bacillus subtilis was isolated from both the cerebrospinal fluid and blood. She was cured by the administration of vancomycin. Next-generation sequencing identified the strain as B. subtilis var. natto, the same strain found in natto, which this patient ate daily. We suspected that some of the B. subtilis that caused the infection may have actually been B. subtilis var. natto.

With the emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), a high-speed and convenient detection technology should be at the forefront of medical care worldwide. This study evaluated the usefulness of GeneSoC, a compact, high-speed reciprocal flow quantitative reverse transcription polymerase chain reaction system, for the detection of SARS-CoV-2. The results support the use of this system for the rapid identification of SARS-CoV-2. This approach can contribute to the strategic selection of initial management strategies for patients with COVID-19.

Background Capnocytophaga canimorsus is a gram-negative bacterium and an oral commensal in dogs and cats, but occasionally causes serious infections in humans. Septicemia is one of the most fulminant forms, but diagnosis of C. canimorsus infection is often difficult mainly because of its very slow growth. C. canimorsus infective endocarditis (IE) is rare and is poorly understood. Since quite a few strains produce β-lactamase, antimicrobial susceptibility is pivotal information for adequate treatment. We herein report a case with C. canimorsus IE and the results of drug susceptibility test. Case presentation A 46-year-old man had a dog bite in his left hand 3 months previously. The patient was referred to our hospital for fever (body temperature > 38 °C), visual disturbance, and dyspnea. Echocardiography showed aortic valve regurgitation and vegetation on the leaflets. IE was diagnosed, and we initially administered cefazolin and gentamycin assuming frequently encountered microorganisms and the patient underwent aortic valve replacement. C. canimorsus was detected in the aortic valve lesion and blood cultures. It was also identified by 16S ribosome DNA sequencing. Ceftriaxone were started and continued because disk diffusion test revealed the isolate was negative for β-lactamase and this case had cerebral symptoms. The patient successfully completed antibiotic treatment following surgery. Conclusions We diagnosed C. canimorsus sepsis and IE by extended-period blood cultures and 16S ribosome DNA sequencing by polymerase chain reaction, and successfully identified its drug susceptibility.

To assess the time trend of diagnostic accuracy of pre- and post-eradicationA series 100 of conventional endoscopic image sets taken from subjects undergoing gastric cancer screening at a polyclinic were evaluated by 5 experienced assessors. Each assessor independently examined endoscopic findings according to the Kyoto classification and then determined theThe overall diagnostic accuracy ofDiagnostic accuracy of

Purpose . The new third-generation sequencing platform MinION is an attractive maintenance-free and disposable portable tool that can perform long-read and real-time sequencing. In this study, we validated this technology for the identification of pathogens from positive blood culture (BC) bottles. Methodology . A total of 38 positive BC bottles were collected from patients with bloodstream infections, and 18 isolates of Gram-negative (GN) bacteria and 20 isolates of Gram-positive (GP) bacteria were identified from these using 16S rRNA sequencing and then used in this study. DNA was extracted from each aliquot using an extraction protocol that combined glass bead beating and chemical lysis. Up to 200 ng of each purified DNA sample was processed for library preparation and whole-genome sequencing was performed on up to 12 samples through a single MinION flow cell. Results . All GN bacteria identifications made by MinION sequencing for 30 min using the What’s In My Pot? (WIMP) workflow via EPI2ME on the basis of the most frequent classified reads were consistent with those made by 16S rRNA sequencing. On the other hand, for GP bacteria specimens, the identification results for 16S rRNA sequencing and MinION were only in agreement in 12 out of 20 (60.0 %) cases. ARMA analysis was able to detect extended-spectrum β-lactamase (ESBL)-associated genes among various antimicrobial resistance-related genes. Conclusion . We demonstrated the potential of the MinION sequencer for the identification of GN bacteria from positive BC bottles and the confirmation of an ESBL phenotype. This innovative sequence technology and its application could lead to a breakthrough in the diagnosis of infectious diseases.

Purpose. Bloodstream infections are major causes of morbidity and mortality that lead to prolonged hospital stays and higher medical costs. In this study, we aimed to evaluate the MinION nanopore sequencer for the identification of the most dominant pathogens in positive blood culture bottles. Methodology. 16S and ITS1-5.8S-ITS2 rRNA genes were amplified by PCR reactions with barcoded primers using nine clinical isolates obtained from positive blood bottles and 11 type strains, including five types of Candida species. Barcoded amplicons were mixed, and multiplex sequencing with the MinION sequencer was performed. In addition, barcoded PCR amplicons were sequenced by Sanger sequencing to validate the performance of the MinION. Results. The bacterial and Candida spp. identified by MinION sequencing, based on the highest homology of reference sequences from the NCBI gene databases, agreed with the matrix-assisted laser desorption ionization time of flight mass spectrometry results, excepting the closely related species Streptococcusand Escherichia coli. The ‘pass’ reads obtained within about 10 min of sequencing were sufficient to identify the pathogens. The average values of sequence identities with 1D2 chemistry and the R9.5 flow cell were around 99 %; thus, frequent sequence errors did not affect species identification based on amplicon sequencing. Conclusion. We have established a rapid, portable and economical technique for the identification of pathogens in positive blood culture bottles through a novel MinION nanopore sequencer amplicon sequencing scheme, which replaces traditional Sanger sequencing.

Background Klebsiella variicola and K. quasipneumoniae are new species distinguishable from K. pneumoniae but they are often misidentified as K. pneumoniae in clinical settings. Several reports have demonstrated the possibility that the virulence factors and clinical features differ among these three phylogroups. In this study, we aimed to clarify whether there were differences in clinical and bacterial features between the three phylogroups isolated from patients with bloodstream infections (BSIs) in Japan. Methods Isolates from all patients with BSIs caused by K. pneumoniae admitted to two hospitals between 2014 and 2017 (n = 119) were included in the study. Bacterial species were identified via sequence analysis, and their virulence factors and serotypes were analyzed via multiplex PCR results. Clinical data were retrieved from medical records. Results Of the 119 isolates, 21 (17.7%) were identified as K. variicola and 11 (9.2%) as K. quasipneumoniae; K1 serotype was found in 16 (13.4%), and K2 serotype in 13 (10.9%). Significant differences in the prevalence of rmpA, iutA, ybtS, entB and kfu (p allS genes (p K. variicola was more frequently detected in patients over 80 years old compared with other Klebsiella species (p K. quasipneumoniae more frequently occurred in patients with malignancy (p Conclusions Our findings demonstrated the differences in bacterial pathogenicity and clinical features among these three phylogroups. Further epidemiological studies into BSI caused by Klebsiella species are warranted.

Background and Aim: Reddish depressed lesions (RDLs) frequently observed in patients following Helicobacter pylori eradication are indistinguishable from gastric cancer. We examined the clinical and histological feature of RDLs and its relevant endoscopic diagnosis including magnifying narrow-band imaging (M-NBI). Methods: We enrolled 301 consecutive patients with H. pylori eradication who underwent endoscopy using white light imaging (WLI). We examined the prevalence and host factors contributing to the presence of RDLs. Next, we used M-NBI in 90 patients (104 RDLs), and compared the diagnostic efficacy between M-NBI and WLI groups using propensity-score matching analysis. Results: In 301 patients after eradication, 117 (39%) showed RDLs. Male, open-type atrophy, and gastric cancer history were risk factors for RDLs. A gastric biopsy was needed in 83 (71%) during WLI observation and only 2 were diagnosed with adenocarcinoma. In M-NBI group, a biopsy was performed in 21 (20%), and 9 were diagnosed with adenocarcinoma. A biopsy was required in fewer patients, and the positive predictive value of a biopsy was statistically higher in M-NBI than in the WLI group (p < 0.01). Conclusions: RDLs are frequently observed in high-risk patients for gastric cancer after eradication. M-NBI demonstrated significantly superior diagnostic efficacy with respect to RDL.

Background and aims Pathogenic auto-antibodies produced by plasma cells are key drivers of many auto-immune diseases such as Systemic Lupus Erythematosus (SLE), Rheumatoid Arthritis (RA), and Sjogren’s Syndrome (SS). In addition, solid organ transplant rejection is also mediated by antibodies produced against the donor organ. Plasma cells are highly metabolically active antibody factories and thus sensitive to depletion by proteasome inhibitors. Ixazomib, an oral proteasome inhibitor, was recently approved in the US and Canada for use in combination with lenalidomide and dexamethasone in patients with multiple myeloma who have received at least 1 prior therapy. The Keyhole limpet hemocyanin (KLH) model of T cell-dependent antigen response was used to determine if ixazomib depletes plasma cells resulting in a reduction of antibodies. Methods Briefly, rats were immunised with KLH and TiterMax adjuvant then treated with ixazomib twice weekly until study termination. Results Treatment with ixazomib significantly inhibited anti-KLH antibodies by 34% (p Conclusions Ixazomib depleted plasma cells resulting in reduced antibodies suggesting further preclinical studies are warranted in diseases with pathogenic antibodies such as SLE, RA, SS and solid organ transplant rejection.

Background and aims Auto-antibodies to nuclear constituents and type I Interferons (IFN) such as IFN-a play key roles in pathogenesis of Systemic Lupus Erythematosus (SLE). Ixazomib, an oral proteasome inhibitor, approved in the US and Canada for use in combination with lenalidomide and dexamethasone in patients with multiple myeloma who have received at least 1 prior therapy. Proteasome inhibitors like ixazomib that may deplete plasma cells and cellular sources of IFN-a are also attractive for autoimmune diseases like SLE. To investigate the potential of ixazomib the MRL/lpr model was used as it has extensively been shown to replicate many features of SLE. Methods MRL/lpr animals received oral ixazomib twice a week for 4 weeks. Results Ixazomib suppressed the time-dependent increase in anti-dsDNA IgG antibodies, resulting in 73% (p Conclusions These findings suggest that ixazomib may be an effective agent for treating antibody-mediated diseases such as SLE by depleting both plasma cells the source of pathogenic antibodies and pDCs the main source of type I IFN production. An ongoing randomised, double-blind phase Ib study is investigating multiple rising doses of Ixazomib (MLN9708) for the treatment of patients with ISN/RPS class III, IV or V lupus nephritis who have not responded adequately to current therapy.

Adipocytes accumulated in the visceral area change their function to induce tumor necrosis factor-alpha (TNF-alpha) secretion with concomitant matrix metalloproteinase (MMP)-3 induction in mice. This study was performed to clarify the role of macrophages (Mphi)-secreted MMP on the functional changes in adipocytes using a culture system.Cultures of 3T3-L1 adipocytes with THP-1 Mphi or the Mphi-conditioned medium were used to investigate the role of Mphi-MMP on the TNF-alpha gene in 3T3-L1 adipocytes by the addition of MMP inhibitors. For animal experiments, male C57BL/6J mice were rendered insulin resistant by feeding a high-fat diet, and the expression of an Mphi marker F4/80, and MMP-3 genes in mesenteric and subcutaneous fat tissue specimens were examined.Mphi-conditioned media (Mphi-CM) increased the levels of TNF-alpha mRNA expression in 3T3-L1 adipocytes, and these adipocyte responses were abolished by treatment with GM6001, a broad-spectrum MMP inhibitor, or NNGH (N-isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid), an MMP-3 inhibitor. The activated form of MMP-3 enhanced glycerol release as well as TNF-alpha protein secretion from 3T3-L1 adipocytes. The incubation of adipocytes with MMP-3 inhibited insulin-induced glucose uptake in adipocytes. Furthermore, a high-fat intake increased the expression of MMP-3, decreased the insulin-induced glucose uptake of adipocytes and induced expression of F4/80 in mesenteric fat tissue of C57BL/6 mice.Mphi may cause a pathological link with surrounding adipocytes through the secretion of MMP-3 followed by TNF-alpha expression in adipocytes in visceral fat tissue.

Summary: Chronic hypertension is associated with structural and functional changes in the cerebrovascular bed, which influence cerebral circulation and its autoregulation. We examined whether or not long-term treatment of spontaneously hypertensive rats (SHRs) with the new calcium antagonist AE0047 would reverse structural changes in the cerebral vasculature and normalize the elevated lower blood pressure (BP) limit of the cerebral blood flow (CBF) autoregulation. Treating 6-month-old SHRs with a diet containing either 0.013 or 0.04% AE0047 for 8 weeks reduced BP and, at the higher dose, maintained BP at a level similar to that of age-matched Wistar-Kyoto (WKY) rats. At the end of the treatment period, CBF was measured by using the hydrogen- clearance method, and the lower limit of the autoregulation curve was estimated by repeated CBF measurement with stepwise reduction of BP through bleeding. This limit was significantly higher in untreated SHRs than in WKY rats (111 ± 8 vs. 60 ± 8 mm Hg). AE0047 caused a significant and dose-dependent shift in the elevated lower BP limit, which decreased to 83 ± 9 and 75 ± 6 mm Hg at the low and high dose, respectively. In perfusion-fixed proximal, intermediate, and distal portions of the middle cerebral artery, media thickness/external diameter (M/ED) ratios were significantly greater in untreated SHRs than in WKY rats. In AE0047-treated animals, M/ED ratios in all portions tended to be reduced halfway between those for untreated SHRs and those for WKY rats, but with no statistical significance. These results suggest that long-term treatment of patients with hypertension with AE0047 will normalize the autoregulatory threshold while preserving CBF and thereby improve tolerance to BP reduction, but the potential to ameliorate structural alterations may be small.

AE0047 [4-(4-benzhydrylpiperazino)phenethyl methyl 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridine dicarboxylate dihydrochloride] is a new dihydropyridine calcium antagonist with protective effects against cerebral ischaemia and the occurrence of stroke in several animal models. We investigated the effects of AE0047 on focal ischaemia induced by middle cerebral artery occlusion in stroke-prone spontaneously hypertensive rats. AE0047 at a dose causing 20 or 40% systemic hypotension (1 or 3 mg kg−) was given orally twice, 15 min and 24 h after occlusion. The neurological status of animals was investigated 2, 24 and 48 h after occlusion. Infarct area of brain was measured 48 h after occlusion. Middle cerebral artery occlusion resulted in the progressive deterioration of neurological status and large infarction in middle cerebral artery territories with 40% mortality. AE0047 dose-dependently attenuated the deterioration of neurological status, and reduced mortality to 0 or 10%. AE0047 significantly reduced infarct size and left/right hemispheric area ratio, an index of brain swelling. These results suggest that AE0047 has the ability to ameliorate ischaemic cerebral stroke in hypertensive patients.

Chronic pressure overload induces cardiac tissue remodeling. Chymase is known to regulate matrix metabolism and angiotensin II formation. In the present study, we investigated the pathophysiological functions of chymase in the pressure-overloaded hamster heart induced by a two-kidney, one-clip (2K1C) hypertension procedure. Fibrosis and apoptosis were observed in the pressure-overloaded hearts of 2K1C hamsters 32 weeks after clipping, but these histological changes were not detected at 16 weeks. Heart chymase-like activity of 2K1C hamsters at 32 weeks increased 5.2-fold compared with that at 16 weeks, while angiotensin-converting enzyme was not activated. Chymase might be involved in cardiac tissue remodeling during the chronic stage of hypertension.