Incidence of and risk factors for suspected COVID-19 reinfection in Kyoto City: a population-based epidemiological study.

To determine the clinical characteristics of and risk factors for suspected reinfection with coronavirus 2019 (COVID-19). This was a retrospective cohort study using population-based notification records of residents in Kyoto City (1.4 M) with laboratory-confirmed COVID-19 infection between 1 March 2020 and 15 April 2022. Reinfection was defined by two or more positive COVID-19 test results ≧ 90 days apart. Demographic characteristics, the route and timing of infection and history of vaccination were analysed to identify risk factors for reinfection. Among the cohort of 107,475 patients, reinfection was identified in 0.66% (n = 709). The age group with the highest reinfection rate was 18-39 years (1.06%), followed by 40-59 years (0.58%). Compared to the medical and nursing professionals, individuals who worked in the construction and manufacturing industry (odds ratio [OR]: 2.86; 95% confidence interval [CI]: 1.66-4.92) and hospitality industry (OR: 2.05; 95% CI: 1.28-.31) were more likely to be reinfected. Symptomatic cases at initial infection, receiving more than 2 doses of vaccination and risk factors for severe infection at initial infection were protective factors against reinfection. Of the reinfected individuals, the reinfection route was unknown in 65%. Reinfection with COVID-19 is uncommon, with suspected reinfections more likely in adults, those with high exposure and unvaccinated individuals; the reinfection route was unknown in the majority of cases. This study confirmed the need to continue with self-protection efforts and to implement vaccination programs in high-risk populations.

A population-based study of the trend in SARS-CoV-2 diagnostic modalities from the beginning of the pandemic to the Omicron surge in Kyoto City, Kyoto, Japan.

BACKGROUND: The coronavirus disease 2019 (COVID-19) presents critical diagnostic challenges for managing the pandemic. We investigated the 30-month changes in COVID-19 testing modalities and functional testing sites from the early period of the pandemic to the most recent Omicron surge in 2022 in Kyoto City, Japan. METHODS: This is a retrospective-observational study using a local anonymized population database that included patients' demographic and clinical information, testing methods and facilities from January 2020 to June 2022, a total of 30 months. We computed the distribution of symptomatic presentation, testing methods, and testing facilities among cases. Differences over time were tested using chi-square tests of independence. RESULTS: During the study period, 133,115 confirmed COVID-19 cases were reported, of which 90.9% were symptomatic. Although nucleic acid amplification testing occupied 68.9% of all testing, the ratio of lateral flow devices (LFDs) rapidly increased in 2022. As the pandemic continued, the testing capability was shifted from COVID-19 designated facilities to general practitioners, who became the leading testing providers (57.3% of 99,945 tests in 2022). CONCLUSIONS: There was a dynamic shift in testing modality during the first 30 months of the pandemic in Kyoto City. General practitioners increased their role substantially as the use of LFDs spread dramatically in 2022. By comprehending and documenting the evolution of testing methods and testing locations, it is anticipated that this will contribute to the establishment of an even more efficient testing infrastructure for the next pandemic.

Chromatographic purification of small extracellular vesicles using an affinity column for phospholipid membranes.

OBJECTIVES: This study aimed to investigate whether chromatography using an ExoPUA column, an affinity column for phospholipid membranes, could potentially serve as an efficient, rapid, scalable, and reproducible method for purifying small extracellular vesicles (sEVs). RESULTS: We used the ExoPUA column connected to a fast-performance liquid chromatography system. One-step chromatographic purification of sEVs from culture supernatant using the ExoPUA protocol resulted in an 82 ± 16-fold increase in purity with a yield of 38 ± 5% of sEVs. The purified sEVs contained CD9, CD63, TSG101, and miRNA (miR-21), but not the endoplasmic reticulum protein Calnexin. Transmission electron microscopy indicated that the purified sEVs were intact. The purification performance of the ExoPUA protocol showed superior results in terms of yield compared to that of the differential ultracentrifugation method, the most commonly used method for purifying sEVs in laboratories, and purity compared to that of the DEAE chromatography protocol. CONCLUSION: The sEVs were effectively purified in the bind-elute mode and the ExoPUA column can be refreshed and sterilized with sodium hydroxide (NaOH), having high potential for multiple sEV purification in a scalable and industrial manner.

Clinical outcomes after tracheostomy in patients with coronavirus disease 2019: a single-center experience in Japan.

PURPOSE: Many patients with coronavirus disease 2019 require mechanical ventilation and tracheostomy. However, the timing and indications for tracheostomy are controversial. This study assessed 11 patients with coronavirus disease 2019 who underwent tracheostomy with clinical information and retrospective analyses. METHODS: A single-center retrospective observational study was performed on patients with coronavirus disease 2019 who underwent tracheostomy between 2020 and 2021. RESULTS: Failure to wean was the most common indication for tracheostomy, followed by extracorporeal membrane oxygenation decannulation and the need for secretion management. After tracheostomy, six patients (54.5%) were liberated from the ventilator. The time from intubation to tracheostomy (21.1 ± 9.14 days) was correlated with the duration of ventilator dependency (36.83 ± 20.45 days, r2 = 0.792, p = 0.018). The mean Acute Physiological and Chronic Health Evaluation II score was significantly lower in the ventilator-liberated group (23 ± 2.77) than in the non-ventilator-liberated group (31 ± 6.13, p = 0.0292). Furthermore, patients with Acute Physiological and Chronic Health Evaluation II scores of < 27 points achieved ventilator liberation and a long-term survival (p = 0.0006). CONCLUSIONS: This study describes the outcomes of a cohort of patients who underwent tracheostomy after intubation for coronavirus disease 2019. The Acute Physiological and Chronic Health Evaluation II score predicted whether or not the patient could achieve ventilator liberation.

Transmissibility of SARS-CoV-2 B.1.1.214 and Alpha Variants during 4 COVID-19 Waves, Kyoto, Japan, January 2020-June 2021.

Household transmission is a primary source of SARS-CoV-2 spread. We used COVID-19 epidemiologic investigation data and viral genome analysis data collected in the city of Kyoto, Japan, during January 2020-June 2021 to evaluate the effects of different settings and viral strains on SARS-CoV-2 transmission. Epidemiologic investigations of 5,061 COVID-19 cases found that the most common category for close contact was within households (35.3%); this category also had the highest reverse transcription PCR positivity. The prevalent viral lineage shifted from B.1.1.214 in the third wave to the Alpha variant in the fourth wave. The proportion of secondary cases associated with households also increased from the third to fourth waves (27% vs. 29%). Among 564 contacts from 206 households, Alpha variant was significantly associated with household transmission (odds ratio 1.52, 95% CI 1.06-2.18) compared with B.1.1.214. Public health interventions targeting household contacts and specific variants could help control SARS-CoV-2 transmission.

A preliminary pilot study investigating the impact of endovascular treatment on leg muscle volume in peripheral artery disease and its relation to baseline glycemic control.

BACKGROUND AND AIMS: Peripheral artery disease (PAD), intermittent claudication, and impaired mobility contribute to the loss of skeletal muscle. This study investigated the impact of endovascular treatment (EVT) in patients suffering from PAD above the knee and its relation to baseline glycemic control. METHODS AND RESULTS: Mid-thigh muscle volume was measured before EVT, 3 months after EVT and 6 months after EVT. Mid-thigh muscle volumes of ipsilateral PAD patients with ischemic and non-ischemic legs were compared. Correlations between total thigh muscle volume and clinical characteristics were analyzed using univariable and multivariable analysis. Overall, thigh muscle volume increased after EVT. The mid-thigh muscle volume was significantly lower in patients with ipsilateral lesions and in those with ischemic lower limbs. The thigh muscle volume of those with ischemic lower limbs increased after EVT. Baseline glycated hemoglobin was the only factor that was negatively correlated with changes in the muscle volume after EVT. Muscle volume significantly increased in normoglycemic HbA1c<6.5% (47 mmol/mol) patients. There was no significant alteration in the muscle volume of hyperglycemic HbA1c ≥ 6.5% patients. CONCLUSION: Ischemic muscle atrophy was ameliorated after EVT in normoglycemic patients. There is a need for a large-scale trial to investigate whether EVT can protect or delay skeletal muscle loss.

Bacterial biosilicification: a new insight into the global silicon cycle.

Biosilicification is the process by which organisms incorporate soluble, monomeric silicic acid, Si(OH)4, in the form of polymerized insoluble silica, SiO2. Biosilicifying eukaryotes, including diatoms, siliceous sponges, and higher plants, have been the targets of intense research to study the molecular mechanisms underlying biosilicification. By contrast, prokaryotic biosilicification has been less well studied, partly because the biosilicifying capability of well-known bacteria was not recognized until recently. This review summarizes recent findings on bacterial extracellular and intracellular biosilicification, the latter of which has been demonstrated only recently in bacteria. The topics discussed herein include bacterial (and archaeal) extracellular biosilicification in geothermal environments, encapsulation of Bacillus spores within a silica layer, and silicon accumulation in marine cyanobacteria. The possible contribution of bacterial biosilicification to the global silicon cycle is also discussed.

Insulin sensor cells for the analysis of insulin secretion responses in single living pancreatic ß cells.

Investigation of the functions of insulin-secreting cells in response to glucose in single-living cells is essential for improving our knowledge on the pathogenesis of diabetes. Therefore, it is desired to develop a new convenient method that enables the direct detection of insulin secreted from single-living cells. Here, insulin-sensor-cells expressing a protein-based insulin-detecting probe immobilized on the extracellular membrane were developed to evaluate the insulin-secretion response in single-living pancreatic ß cells. The protein-based insulin-detecting probe (NαLY) was composed of a bioluminescent protein (nano-luc), the αCT segment of the insulin receptor, L1 and CR domains of the insulin receptor, and a fluorescent protein (YPet). NαLY exhibited a bioluminescence resonance energy transfer (BRET) signal in response to insulin; thus, cells of Hepa1-6 line were genetically engineered to express NαLY on the extracellular membrane. The cells were found to act as insulin-sensor-cells, exhibiting a BRET signal in response to insulin. When the insulin-sensor-cells and pancreatic ß cells (MIN6 cell line) were cocultured and stimulated with glucose, insulin-sensor-cells nearby pancreatic ß cells showed the spike-shaped BRET signal response, whereas the insulin-sensor-cells close to one pancreatic ß cell did not exhibit such signal response. However, all the insulin-sensor-cells showed a gradual increase in BRET signals, which were presumably attributed to the increase in insulin concentrations in the culture dish, confirming the function of these insulin-sensor-cells. Therefore, we demonstrated that heterogenetic insulin secretion in single-living pancreatic ß cells could be measured directly using the insulin sensor cells.

Arginine-mediated dissociation of single cells and cell sheets from a polystyrene culture dish.

Here, we report a novel non-enzymatic cell dissociation method, based on our finding that adherent cells dissociate rapidly from the polystyrene culture dish when incubated in an l- or d-arginine-containing solution. We also demonstrate the successful detachment of confluent NIH/3T3 cell monolayers from the culture dish as a cell sheet by the addition of an arginine solution.

Pharmacokinetics of insulin disappearance after massive overdosing.

Long-term glucose supplementation is required to prevent hypoglycemia after massive insulin overdosing. We fitted the blood insulin concentration-time profile to the model: I = A·exp(-a·t) + B·exp(-b·t), where I (µU/mL) is the serum/plasma insulin concentration, A (µU/mL) and B (µU/mL) are the peak insulin concentrations of each component, a (time-1) and b (time-1) are the time constants of each component, and t (h) is the time elapsed from the peak of blood insulin level. Additional components were considered as needed. Patient 1 had auto-injected 600 U NovoRapid® 30Mix, and Patient 2 had auto-injected 300 U Novolet®R (regular) and 1,800 U NovoLet®N (NPH). We used the disappearance of therapeutic doses of the respective insulin in healthy individuals as controls, and we obtained parameters by Excel solver. In Patient 1, the parameter values were A = 1490.04 and a = 0.15 for insulin aspart and B = 60.66 and b = 0.04 for protaminated aspart. In Patient 2, the values were A = 784.45 and a = 0.38 for regular insulin and B = 395.84 and b = 0.03 for NPH. Compared with controls, the half-lives (t1/2) for insulin aspart and protaminated aspart were 4 and 2 times longer, respectively, in Patient 1. In Patient 2, the t1/2 for regular and NPH insulin were 2 and 7 times longer than those in the controls, respectively. In conclusion, the t1/2 for insulin was elongated 2 to 7 times after massive overdosing, explaining why glucose supplementation is needed for long periods in these cases.

The C-Terminal Zwitterionic Sequence of CotB1 Is Essential for Biosilicification of the Bacillus cereus Spore Coat.

UNLABELLED: Silica is deposited in and around the spore coat layer of Bacillus cereus, and enhances the spore's acid resistance. Several peptides and proteins, including diatom silaffin and silacidin peptides, are involved in eukaryotic silica biomineralization (biosilicification). Homologous sequence search revealed a silacidin-like sequence in the C-terminal region of CotB1, a spore coat protein of B. cereus. The negatively charged silacidin-like sequence is followed by a positively charged arginine-rich sequence of 14 amino acids, which is remarkably similar to the silaffins. These sequences impart a zwitterionic character to the C terminus of CotB1. Interestingly, the cotB1 gene appears to form a bicistronic operon with its paralog, cotB2, the product of which, however, lacks the C-terminal zwitterionic sequence. A ΔcotB1B2 mutant strain grew as fast and formed spores at the same rate as wild-type bacteria but did not show biosilicification. Complementation analysis showed that CotB1, but neither CotB2 nor C-terminally truncated mutants of CotB1, could restore the biosilicification activity in the ΔcotB1B2 mutant, suggesting that the C-terminal zwitterionic sequence of CotB1 is essential for the process. We found that the kinetics of CotB1 expression, as well as its localization, correlated well with the time course of biosilicification and the location of the deposited silica. To our knowledge, this is the first report of a protein directly involved in prokaryotic biosilicification. IMPORTANCE: Biosilicification is the process by which organisms incorporate soluble silicate in the form of insoluble silica. Although the mechanisms underlying eukaryotic biosilicification have been intensively investigated, prokaryotic biosilicification was not studied until recently. We previously demonstrated that biosilicification occurs in Bacillus cereus and its close relatives, and that silica is deposited in and around a spore coat layer as a protective coating against acid. The present study reveals that a B. cereus spore coat protein, CotB1, which carried a C-terminal zwitterionic sequence, is essential for biosilicification. Our results provide the first insight into mechanisms required for biosilicification in prokaryotes.

Continuous Monitoring of Specific mRNA Expression Responses with a Fluorescence Resonance Energy Transfer-Based DNA Nano-tweezer Technique That Does Not Require Gene Recombination.

This letter discusses the feasibility of continuously monitoring specific mRNA expression responses in a living cell with a probe structured as a fluorescence resonance energy transfer (FRET)-based DNA nano-tweezer (DNA-NT). The FRET-based DNA-NT, self-assembled from three single-stranded DNAs, alters its structure from an open state to a closed state in recognition of a target mRNA, resulting in the closing of the distal relation of previously modified FRET-paired fluorescent dyes and generating a FRET signal. The expressions of glucose transporters (GLUT) 1 and 4 in a mouse hepato-carcinoma (Hepa 1-6 cells) were selected as the target model. Live-cell imaging analysis of Hepa 1-6 cells with both FRET-based DNA-NTs indicated that the behaviors of the FRET signals integrated in each individual cell were similar to those measured with the conventional mass analysis technique of semiquantitative real-time (RT) polymerase chain reaction (PCR). From these results, it is concluded that continuous monitoring of gene expression response without gene recombination is feasible with a FRET-based DNA-NT, even in a single cell manner.

Differential Counting of Asbestos Using Phase Contrast and Fluorescence Microscopy.

Considering the increasing use of various asbestos substitutes, asbestos risk management in many industries may require accurate techniques for detecting and distinguishing asbestos from non-asbestos fibers. Using fluorescently labeled asbestos-binding proteins, we previously developed a novel method for detection and counting of asbestos fibers under fluorescence microscopy (FM). This method can provide speedy, on-site detection and identification of the asbestos fibers and has higher sensitivity than phase contrast microscopy (PCM). However, current asbestos exposure limits are derived from risk assessments based on epidemiological studies that were conducted using PCM fiber counts. Therefore, the sensitivity of asbestos testing should be maintained at PCM level to properly assess compliance with these limit values. Here, we developed and tested a novel application of FM as a differential counting method that complements PCM analysis and is fully compatible with the PCM-based epidemiological data. In the combined PCM-FM method, the fluorescent asbestos-binding probe is applied prior to filter clearing. The method makes it possible to easily switch between two microscopic techniques while analyzing the same fields of view: PCM is used for counting fibers, and FM for differentiating asbestos from non-asbestos fibers. Using airborne dust samples from demolition sites in Japan, we compared PCM-FM with scanning electron microscopy (SEM)-based differential counting method. Statistical analysis indicated a slight conservative bias of PCM-FM method, combined with relatively high variability across the full range of fiber concentrations in our sample set. Using correlative microscopy, we also evaluated the specificity of FM staining, which is a potential cause of variability between the two methods. The energy-dispersive X-ray analysis indicated that ~95% of fluorescently stained fibers in the demolition site samples were correctly identified as asbestos. While further research is needed to fully clarify the causes of variability between FM- and SEM-based differential counting, PCM-FM could be used for rapid and selective detection of asbestos fibers in field samples.

A BRET-based homogeneous insulin assay using interacting domains in the primary binding site of the insulin receptor.

A new homogeneous insulin assay requiring no chemical modification of an insulin recognition domain, which can be applied to continuous monitoring of the time-dependent cellular response in vitro, was developed. The carboxy-terminal α-chain (αCT) segment and first leucine-rich-repeat (L1) domain in the primary binding site on the insulin receptor were genetically fused with a bioluminescent protein (Nanoluc, Nluc) and a fluorescent protein (yellow fluorescent protein, YPet) to produce the insulin-sensing probe proteins Nluc-αCT and L1-YPet. The BRET signal was observed on simple mixing of insulin with these protein probes, in a so-called homogeneous assay. The BRET signal was proportional to the insulin concentration, and the lower detection limit was 0.8 µM. Time-dependent insulin secretion from drug-stimulated MIN6 cells was also successfully monitored continuously with the probe proteins. This BRET-based homogeneous insulin assay method is thus expected to be applicable to drug development by high-throughput screening.

Development of an automated asbestos counting software based on fluorescence microscopy.

An emerging alternative to the commonly used analytical methods for asbestos analysis is fluorescence microscopy (FM), which relies on highly specific asbestos-binding probes to distinguish asbestos from interfering non-asbestos fibers. However, all types of microscopic asbestos analysis require laborious examination of large number of fields of view and are prone to subjective errors and large variability between asbestos counts by different analysts and laboratories. A possible solution to these problems is automated counting of asbestos fibers by image analysis software, which would lower the cost and increase the reliability of asbestos testing. This study seeks to develop a fiber recognition and counting software for FM-based asbestos analysis. We discuss the main features of the developed software and the results of its testing. Software testing showed good correlation between automated and manual counts for the samples with medium and high fiber concentrations. At low fiber concentrations, the automated counts were less accurate, leading us to implement correction mode for automated counts. While the full automation of asbestos analysis would require further improvements in accuracy of fiber identification, the developed software could already assist professional asbestos analysts and record detailed fiber dimensions for the use in epidemiological research.

A new subfamily of polyphosphate kinase 2 (class III PPK2) catalyzes both nucleoside monophosphate phosphorylation and nucleoside diphosphate phosphorylation.

Inorganic polyphosphate (polyP) is a linear polymer of tens to hundreds of phosphate (Pi) residues linked by "high-energy" phosphoanhydride bonds as in ATP. PolyP kinases, responsible for the synthesis and utilization of polyP, are divided into two families (PPK1 and PPK2) due to differences in amino acid sequence and kinetic properties. PPK2 catalyzes preferentially polyP-driven nucleotide phosphorylation (utilization of polyP), which is important for the survival of microbial cells under conditions of stress or pathogenesis. Phylogenetic analysis suggested that the PPK2 family could be divided into three subfamilies (classes I, II, and III). Class I and II PPK2s catalyze nucleoside diphosphate and nucleoside monophosphate phosphorylation, respectively. Here, we demonstrated that class III PPK2 catalyzes both nucleoside monophosphate and nucleoside diphosphate phosphorylation, thereby enabling us to synthesize ATP from AMP by a single enzyme. Moreover, class III PPK2 showed broad substrate specificity over purine and pyrimidine bases. This is the first demonstration that class III PPK2 possesses both class I and II activities.

Affinity purification of recombinant proteins using a novel silica-binding peptide as a fusion tag.

We recently reported that silica is deposited on the coat of Bacillus cereus spores as a layer of nanometer-sized particles (Hirota et al. 2010 J Bacteriol 192: 111-116). Gene disruption analysis revealed that the spore coat protein CotB1 mediates the accumulation of silica (our unpublished results). Here, we report that B. cereus CotB1 (171 amino acids [aa]) and its C-terminal 14-aa region (corresponding to residues 158-171, designated CotB1p) show strong affinity for silica particles, with dissociation constants at pH 8.0 of 2.09 and 1.24 nM, respectively. Using CotB1 and CotB1p as silica-binding tags, we developed a silica-based affinity purification method in which silica particles are used as an adsorbent for CotB1/CotB1p fusion proteins. Small ubiquitin-like modifier (SUMO) technology was employed to release the target proteins from the adsorbed fusion proteins. SUMO-protease-mediated site-specific cleavage at the C-terminus of the fused SUMO sequence released the tagless target proteins into the liquid phase while leaving the tag region still bound to the solid phase. Using the fluorescent protein mCherry as a model, our purification method achieved 85 % recovery, with a purity of 95 % and yields of 0.60 ± 0.06 and 1.13 ± 0.13 mg per 10-mL bacterial culture for the CotB1-SUMO-mCherry and CotB1p-SUMO-mCherry fusions, respectively. CotB1p, a short 14-aa peptide, which demonstrates high affinity for silica, could be a promising fusion tag for both affinity purification and enzyme immobilization on silica supports.

Exploring Possible Links: Thigh Muscle Mass, Apolipoproteins, and Glucose Metabolism in Peripheral Artery Disease-Insights from a Pilot Sub-Study following Endovascular Treatment.

Peripheral artery disease (PAD) compromises walking and physical activity, which results in further loss of skeletal muscle. The cross-sectional area of the thigh muscle has been shown to be correlated with systemic skeletal muscle volume. In our previous pilot study, we observed an increase in thigh muscle mass following endovascular treatment (EVT) in patients with proximal vascular lesions affecting the aortoiliac and femoropopliteal arteries. Considering the potential interactions between skeletal muscle, lipid profile, and glucose metabolism, we aimed to investigate the relationship between thigh muscle mass and apolipoproteins as well as glucose metabolism in PAD patients undergoing EVT. This study is a prespecified sub-study conducted as part of a pilot study. We prospectively enrolled 22 symptomatic patients with peripheral artery disease (PAD) and above-the-knee lesions, specifically involving the blood vessels supplying the thigh muscle. The mid-thigh muscle area was measured with computed tomography before and 6 months after undergoing EVT. Concurrently, we measured levels of apolipoproteins A1 (Apo A1) and B (Apo B), fasting blood glucose, 2 h post-load blood glucose (using a 75 g oral glucose tolerance test), and glycated hemoglobin A1c (HbA1c). Changes in thigh muscle area (delta muscle area: 2.5 ± 8.1 cm2) did not show significant correlations with changes in Apo A1, Apo B, fasting glucose, 2 h post-oral glucose tolerance test blood glucose, HbA1c, or Rutherford classification. However, among patients who experienced an increase in thigh muscle area following EVT (delta muscle area: 8.41 ± 5.93 cm2), there was a significant increase in Apo A1 (pre: 121.8 ± 15.1 mg/dL, 6 months: 136.5 ± 19.5 mg/dL, p < 0.001), while Apo B remained unchanged (pre: 76.4 ± 19.2 mg/dL, 6 months: 80.5 ± 4.9 mg/dL). Additionally, post-oral glucose tolerance test 2 h blood glucose levels showed a decrease (pre: 189.7 ± 67.5 mg/dL, 6 months: 170.6 ± 69.7 mg/dL, p = 0.075). Patients who exhibited an increase in thigh muscle area demonstrated more favorable metabolic changes compared to those with a decrease in thigh muscle area (delta muscle area: -4.67 ± 2.41 cm2). This pilot sub-study provides insights into the effects of EVT on thigh muscle, apolipoproteins, and glucose metabolism in patients with PAD and above-the-knee lesions. Further studies are warranted to validate these findings and establish their clinical significance. The trial was registered on the University Hospital Medical Information Network Clinical Trials Registry (UMIN000047534).

Predicting heart failure symptoms from the apnoea-hypopnoea index determined by full- night polysomnography.

AIMS: Sleep-disordered breathing (SDB) is closely related to cardiovascular diseases. The higher the apnoea-hypopnoea index (AHI), the higher the prevalence of cardiovascular diseases. Despite these findings suggesting a close link between SDB and heart failure, the relationship between the severity of SDB and the onset of heart failure symptoms in individuals without apparent heart failure symptoms (Heart Failure Stage A + B) remains poorly understood. METHODS AND RESULTS: Between December 2010 and June 2017, we conducted full-night polysomnography (PSG) at the Nippon Medical School Chiba Hokusoh Hospital, extracting patients who were at risk of heart failure (Stage A or B in the Heart Failure Guidelines). Using a median cut-off of AHI ≥ 41.6 events/hour, we divided the patients into two groups and examined the composite endpoint of all-cause mortality plus hospitalization due to heart failure as the primary endpoint. We included 230 patients (mean age 63.0 ± 12.5 years, 78.3% males) meeting the selection criteria. When comparing the two groups, those with AHI < 41.6 events/hour (L group, n = 115) and those with AHI ≥ 41.6 events/hour (H group, n = 115), the H group had higher body mass index and higher serum triglyceride, and lower the frequency of acute coronary syndrome and lower estimated glomerular filtration rate than did the L group, but no other patient characteristics differed significantly. The H group had a significantly higher incidence of the composite endpoint than did the L group (10.6% vs. 2.6%, P = 0.027). Factors associated with the composite endpoint were identified through multivariate analyses, with AHI, haemoglobin, and left atrial dimension emerging as significant factors (hazard ratio [HR] = 1.02, 95% confidence interval [CI] = 1.00-1.04, P = 0.024; HR = 0.71, 95% CI = 0.54-0.94, P = 0.017; and HR = 1.10, 95% CI = 1.03-1.18, P = 0.006, respectively). Conversely, the minimum SpO2 during PSG (<80%) was not associated with the composite endpoint. CONCLUSIONS: In patients with SDB who are at risk of heart failure, severe SDB is associated with a high risk of all-cause mortality and the development of heart failure. Additionally, combining cardiac echocardiography and PSG data may improve risk stratification, offering potential assistance for early intervention. Further examination with a validation cohort is necessary.