We developed a new experimental system in pigs to closely evaluate the process by which H2 is absorbed in the lungs, enters the bloodstream, and is distributed, metabolized, and excreted. We inserted and secured catheters into the carotid artery (CA), portal vein (PV), and supra-hepatic inferior vena cava (IVC) to allow repeated blood sampling and performed bilateral thoracotomy to collapse the lungs. Then, using a hydrogen-absorbing alloy canister, we filled the lungs to the maximum inspiratory level with 100% H2. The pig was maintained for 30 seconds without resuming breathing, as if they were holding their breath. We collected blood from the three intravascular catheters after 0, 3, 10, 30, and 60 minutes and measured H2 concentration by gas chromatography. H2 concentration in the CA peaked immediately after breath holding; 3 min later, it dropped to 1/40 of the peak value. Peak H2 concentrations in the PV and IVC were 40% and 14% of that in the CA, respectively. However, H2 concentration decay in the PV and IVC  was slower than in the CA . At 10 min, H2 concentration was significantly higher in venous blood than in arterial blood. At 60 min, H2 was detected in the portal blood at a concentration of 6.9–53 nL/mL higher than at steady state, and in the SVC 14–29 nL/mL higher than at steady state. In contrast, H2 concentration in the CA decreased to steady state levels. This is the first report showing that inhaled H2 is transported to the whole body by advection diffusion and metabolized dynamically.The benefits of inhaling hydrogen gas (H Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: NoReviewer #3: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: NoReviewer #3: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: This study is a non-clinical pharmacokinetic study of hydrogen in pigs. In addition to the reduction of ischemia-reperfusion injury in the emergency room, hydrogen is also expected to be effective in the treatment of pneumonia with COVID-19, which is currently undergoing clinical trials.　Non-clinical pharmacokinetic studies are required to obtain regulatory approval, but no credible experimental results have been reported to date. This study investigates in detail how hydrogen absorbed from the lungs is transported into the body and metabolized. It is a very well-prepared experiment. It is also valuable in terms of non-clinical pharmacokinetic studies in pigs. I'll only mention minor comments.Comments1). As COVID-19 spread across the globe, scientists are hunting for drugs that could be repurposed as a quick way to tackle the disease. Hydrogen is one such candidate. Therefore, please describe in the discussion the possible mechanisms by which hydrogen may be effective in the treatment of COVID-19-related pneumonia.2). Why were blood concentrations monitored at three locations: the carotid artery and portal vein and the supra-hepatic inferior vena cava?Reviewer #2: To the editorAs a versatile medical gas candidate, hydrogen has attracted much attention in Asia, especially in Japan, China, and Korea, and has been subjected to animal experiments and clinical trials. To date, the only studies to examine the distribution of hydrogen in the body after the hydrogen intake have been conducted in rats, which are small animals. It is significant to investigate the pharmacokinetics of hydrogen in pigs, which are large animals with anatomical similarities to humans. The accuracy of the experiment is high and the results are credible. I would like to offer some minor comments.To the authorsThis study examined how hydrogen absorbed from the lungs diffuses throughout the body when pigs are given a single dose of hydrogen inhalation. After collapsing the lungs by opening the chest, the remaining air in the lungs is expelled by hand compression, and then the lungs are inflated with hydrogen to the level of the maximum inspiratory volume. By maintaining this state for a while, the authors create a state in which the maximum possible dose of hydrogen is taken up from the lungs into the blood. Blood samples are drawn from the carotid artery, portal vein, and supra-hepatic IVC to monitor the kinetics of hydrogen concentrations in the blood. The authors conclude that hydrogen gas is transported to each organ in a blood flow-dependent manner, where it is actively metabolized. As a versatile medical gas candidate, hydrogen has attracted much attention in Asia, especially in Japan, China, and Korea, and has been subjected to animal experiments and clinical trials. To date, the only studies to examine the distribution of hydrogen in the body after the hydrogen intake have been conducted in rats, which are small animals. It is significant to investigate the pharmacokinetics of hydrogen in pigs, which are large animals with anatomical similarities to humans. The accuracy of the experiment is high and the results are credible. I would like to offer some minor comments.Comments(1) This paper examines the pharmacokinetics after hydrogen inhalation, but for the general reader, it should touch on the known pharmacology of hydrogen, such as its antioxidant and anti-inflammatory effects.(2) Hydrogen can be taken into the body not only by inhalation, but also by drinking hydrogen-containing water. What is the pharmacokinetics of hydrogen dissolved in orally ingested water, in analogy with the present experimental results?Reviewer #3: In this study, the concentration of hydrogen in the blood was measured at three locations: the carotid artery, the portal vein, and the supra-hepatic inferior inferior vena cava, after a single inhalation of hydrogen gas in pigs. Although many preclinical and clinical trials have shown the therapeutic effect of hydrogen on a variety of conditions involving ischemia-reperfusion injury, information on how ingested hydrogen is distributed throughout the body is scarce. It is significant that the present study proved that hydrogen absorbed from the lungs is transported by advection diffusion in the bloodstream to systemic organs, rather than by simple diffusion. This study is presented by a group that is leading the world in non-clinical and clinical research on hydrogen gas. The surgery is performed by a transplant surgeon with extensive experience in pig surgery. The concentration of hydrogen was measured by gas chromatography with little error between the two measurements, and the accuracy of the data is high. This data is necessary for hydrogen to be approved by the pharmaceutical industry as a medical gas in the future. I'll only mention minor comments.Comment:(1) Why did you insist on single-dose inhalation instead of continuous inhalation?(2) The treatment of pneumonia caused by COVID-19 has started in Wuhan, China. Please describe the expected pharmacological effects of hydrogen in this clinical setting.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  19 May 2020Comments to the AuthorReviewer #1: This study is a non-clinical pharmacokinetic study of hydrogen in pigs. In addition to the reduction of ischemia-reperfusion injury in the emergency room, hydrogen is also expected to be effective in the treatment of pneumonia with COVID-19, which is currently undergoing clinical trials.　Non-clinical pharmacokinetic studies are required to obtain regulatory approval, but no credible experimental results have been reported to date. This study investigates in detail how hydrogen absorbed from the lungs is transported into the body and metabolized. It is a very well-prepared experiment. It is also valuable in terms of non-clinical pharmacokinetic studies in pigs. I'll only mention minor comments.Comments1). As COVID-19 spread across the globe, scientists are hunting for drugs that could be repurposed as a quick way to tackle the disease. Hydrogen is one such candidate. Therefore, please describe in the discussion the possible mechanisms by which hydrogen may be effective in the treatment of COVID-19-related pneumonia.ResponseThank you very much for kind remarks. We, at the Center for Molecular Hydrogen Medicine at Keio University in Tokyo, have demonstrated the therapeutic effects of H2 on diseases such as acute myocardial infarction , post-cardiac arrest syndrome , hemorrhagic shock , and organ transplantation [10] in both animal experiments and clinical studies. In patients with severe COVID-19, the immune over-response causes the production of large amounts of cytokines by alveolar macrophages, which becomes a cytokine storm, resulting in the progression of acute respiratory distress syndrome, abnormal blood coagulation, and multiple organ failure [21]. H2 gas not only inhibits the overproduction of cytokines [13], but also suppresses vascular endothelial damage [20], facilitates the flow of red blood cells in microvessels and increases the efficiency of gas exchange (M.S. unpublished observation). Accordingly, H2 inhalation therapy has great potential to improve the life expectancy of intubated COVID-19 patients admitted to the intensive care unit with severe hypoxemia. The HYBRID II Trial , a multicenter, randomized, double-blind, placebo-controlled, controlled clinical trial investigating the efficacy of H2 inhalation therapy for patients after out-of-hospital cardiac arrest, has been underway since 2017 using a hydrogenated oxygen supply device jointly developed by Keio University and Taiyo Nippon Sanso (jRCTs031180352)[4]. In the HYBRID II trial, patients after cardiopulmonary arrest and resuscitation have been treated with hydrogenated oxygen for 18 hours in combination with conventional cooling methods. Prior to Hybrid II, we had conducted an open-label, single-arm, prospective interventional trial at Keio University Hospital in Tokyo in 2014 to evaluate the feasibility and safety of H2 inhalation in patients with out-of-hospital cardiac arrest achieved a spontaneous return of circulation [5]. Non-cardiogenic cardiac arrest patients were also enrolled in this TRIAL; 5 patients were entered, one of whom was a CA patient due to severe pneumonia and septic shock. This patient had a stable respiratory state during H2 inhalation, but died after H2 inhalation was completed due to a rapid deterioration of the respiratory state. Based on this experience, we are considering a study protocol in which patients with severe COVID-19 disease will continue to receive hydrogenated oxygen for at least one week until their hypoxemia has sufficiently improved. In China, the hydrogen-oxygen mixture gas inhaler was certified as a national class III medical device in February, and it is reported to have a certain effect on the improvement of hypoxic symptoms of COVID-19-related pneumonia. We hope to bring this new treatment to patients as quickly as possible in order to save the lives of severely ill patients with COVID-19.2). Why were blood concentrations monitored at three locations: the carotid artery and portal vein and the supra-hepatic inferior vena cava?In the Discussion section, I added the reasons why I measured the hydrogen concentration at the three locations as follows.As several clinical studies have been reported or are underway to validate the efficacy of hydrogen inhalation for brain damage, the carotid artery was chosen as the first blood collection point to prove that the inhaled hydrogen can reach the brain efficiently.The liver has a dual blood supply from the portal vein and the hepatic artery. About 75% of the blood flow to the liver comes from the portal vein and 25% from the hepatic artery. Oxygen is supplied by the portal vein and the hepatic artery in half each. Therefore, in contrast to the brain, the liver has been regarded as the organ where inhaled hydrogen is least likely to reach. The liver is the largest organ in the body, performing a number of functions that are essential for life, such as metabolism, detoxification, and excretion, so protecting it with H2 is considered to be a great advantage. Therefore, we wanted to find out how much H2 is consumed as it passes through the liver, so we compared the H2 concentrations in the portal vein and supra-hepatic IVC.Reviewer #2: This study examined how hydrogen absorbed from the lungs diffuses throughout the body when pigs are given a single dose of hydrogen inhalation. After collapsing the lungs by opening the chest, the remaining air in the lungs is expelled by hand compression, and then the lungs are inflated with hydrogen to the level of the maximum inspiratory volume. By maintaining this state for a while, the authors create a state in which the maximum possible dose of hydrogen is taken up from the lungs into the blood. Blood samples are drawn from the carotid artery, portal vein, and supra-hepatic IVC to monitor the kinetics of hydrogen concentrations in the blood. The authors conclude that hydrogen gas is transported to each organ in a blood flow-dependent manner, where it is actively metabolized. As a versatile medical gas candidate, hydrogen has attracted much attention in Asia, especially in Japan, China, and Korea, and has been subjected to animal experiments and clinical trials. To date, the only studies to examine the distribution of hydrogen in the body after the hydrogen intake have been conducted in rats, which are small animals. It is significant to investigate the pharmacokinetics of hydrogen in pigs, which are large animals with anatomical similarities to humans. The accuracy of the experiment is high and the results are credible. I would like to offer some minor comments.Comments(1) This paper examines the pharmacokinetics after hydrogen inhalation, but for the general reader, it should touch on the known pharmacology of hydrogen, such as its antioxidant and anti-inflammatory effects.ResponseThank you very much for summarizing the manuscript and for your positive comments.The biological effects of H2 are explained in relation to the expected therapeutic effects of H2 on COVID-19 in the discussion section.“In patients with severe COVID-19, the immune over-response causes the production of large amounts of cytokines by alveolar macrophages, which becomes a cytokine storm, resulting in the progression of acute respiratory distress syndrome, abnormal blood coagulation, and multiple organ failure [21]. H2 gas not only inhibits the overproduction of cytokines [13], but also suppresses vascular endothelial damage [20], facilitates the flow of red blood cells in microvessels and increases the efficiency of gas exchange (M.S. unpublished observation). Accordingly, H2 inhalation therapy has great potential to improve the life expectancy of intubated COVID-19 patients admitted to the intensive care unit with severe hypoxemia. The HYBRID II Trial , a multicenter, randomized, double-blind, placebo-controlled, controlled clinical trial investigating the efficacy of H2 inhalation therapy for patients after out-of-hospital cardiac arrest, has been underway since 2017 using a hydrogenated oxygen supply device jointly developed by Keio University and Taiyo Nippon Sanso (jRCTs031180352)[4]. In the HYBRID II trial, patients after cardiopulmonary arrest and resuscitation have been treated with hydrogenated oxygen for 18 hours in combination with conventional cooling methods. Prior to Hybrid II, we had conducted an open-label, single-arm, prospective interventional trial at Keio University Hospital in Tokyo in 2014 to evaluate the feasibility and safety of H2 inhalation in patients with out-of-hospital cardiac arrest achieved a spontaneous return of circulation [5]. Non-cardiogenic cardiac arrest patients were also enrolled in this TRIAL; 5 patients were entered, one of whom was a CA patient due to severe pneumonia and septic shock. This patient had a stable respiratory state during H2 inhalation, but died after H2 inhalation was completed due to a rapid deterioration of the respiratory state. Based on this experience, we are considering a study protocol in which patients with severe COVID-19 disease will continue to receive hydrogenated oxygen for at least one week until their hypoxemia has sufficiently improved”. (2) Hydrogen can be taken into the body not only by inhalation, but also by drinking hydrogen-containing water. What is the pharmacokinetics of hydrogen dissolved in orally ingested water, in analogy with the present experimental results?ResponseIf someone drinks H2-rich water, it will be absorbed from the gastrointestinal tract and will first pass through the liver. Our experimental results suggest that most H2 is actively consumed by the liver. Some H2 passes through the liver, but most of it will be discarded in the atmosphere while passing through the lungs. Certainly, the concentration of H2 in the exhaled air after drinking the H2-rich water increases. How much H2 reaches the left ventricle and is supplied to the whole body can be determined by feeding pigs high H2 water and then measuring the H2 concentration in a similar manner.Reviewer #3: In this study, the concentration of hydrogen in the blood was measured at three locations: the carotid artery, the portal vein, and the supra-hepatic inferior inferior vena cava, after a single inhalation of hydrogen gas in pigs. Although many preclinical and clinical trials have shown the therapeutic effect of hydrogen on a variety of conditions involving ischemia-reperfusion injury, information on how ingested hydrogen is distributed throughout the body is scarce. It is significant that the present study proved that hydrogen absorbed from the lungs is transported by advection diffusion in the bloodstream to systemic organs, rather than by simple diffusion. This study is presented by a group that is leading the world in non-clinical and clinical research on hydrogen gas. The surgery is performed by a transplant surgeon with extensive experience in pig surgery. The concentration of hydrogen was measured by gas chromatography with little error between the two measurements, and the accuracy of the data is high. This data is necessary for hydrogen to be approved by the pharmaceutical industry as a medical gas in the future. I'll only mention minor comments.Comment:(1) Why did you insist on single-dose inhalation instead of continuous inhalation?ResponseThank you very much for summarizing the manuscript and for your positive comments. An explanation of why a single inhalation test was performed has been added to the introduction section.“In a non-clinical pharmacokinetic study, the distribution of a test drug to various organs and tissues after a single or repeated dose and its change over time should be investigated. In the case of gas, unlike oral and injectable drugs, a non-clinical pharmacokinetic study with a single dose has not been performed. This was because there was no animal protocol for a single-dose study of the gas. The same is true for H2. The distribution of H2 to various organs and tissues after sustained inhalation has been studied in small animals such as rats. However, it was not possible to determine from these experiments whether H2 diffused from the lungs in a blood flow-independent manner or whether H2 was transported throughout the body in a blood flow-dependent manner. Therefore, in the present study, we devised an animal protocol for single-dose inhalation of gas and proved the latter to be true for the first time”.(2) The treatment of pneumonia caused by COVID-19 has started in Wuhan, China. Please describe the expected pharmacological effects of hydrogen in this clinical setting.The expected pharmacological effects of hydrogen in this clinical setting were described in the discussion section as follows.“In patients with severe COVID-19, the immune over-response causes the production of large amounts of cytokines by alveolar macrophages, which becomes a cytokine storm, resulting in the progression of acute respiratory distress syndrome, abnormal blood coagulation, and multiple organ failure [21]. H2 gas not only inhibits the overproduction of cytokines [13], but also suppresses vascular endothelial damage [20], facilitates the flow of red blood cells in microvessels and increases the efficiency of gas exchange (M.S. unpublished observation). Accordingly, H2 inhalation therapy has great potential to improve the life expectancy of intubated COVID-19 patients admitted to the intensive care unit with severe hypoxemia. The HYBRID II Trial , a multicenter, randomized, double-blind, placebo-controlled, controlled clinical trial investigating the efficacy of H2 inhalation therapy for patients after out-of-hospital cardiac arrest, has been underway since 2017 using a hydrogenated oxygen supply device jointly developed by Keio University and Taiyo Nippon Sanso (jRCTs031180352)[4]. In the HYBRID II trial, patients after cardiopulmonary arrest and resuscitation have been treated with hydrogenated oxygen for 18 hours in combination with conventional cooling methods. Prior to Hybrid II, we had conducted an open-label, single-arm, prospective interventional trial at Keio University Hospital in Tokyo in 2014 to evaluate the feasibility and safety of H2 inhalation in patients with out-of-hospital cardiac arrest achieved a spontaneous return of circulation [5]. Non-cardiogenic cardiac arrest patients were also enrolled in this TRIAL; 5 patients were entered, one of whom was a CA patient due to severe pneumonia and septic shock. This patient had a stable respiratory state during H2 inhalation, but died after H2 inhalation was completed due to a rapid deterioration of the respiratory state. Based on this experience, we are considering a study protocol in which patients with severe COVID-19 disease will continue to receive hydrogenated oxygen for at least one week until their hypoxemia has sufficiently improved”. 1 Jun 2020Pharmacokinetics of a single inhalation of hydrogen gas in pigsPONE-D-20-11712R1Dear Dr. Sano,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: All comments have been addressedReviewer #2: (No Response)Reviewer #3: All comments have been addressed**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: (No Response)Reviewer #3: Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: (No Response)Reviewer #3: Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: (No Response)Reviewer #3: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: (No Response)Reviewer #3: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The authors did a good job addressing the criticisms from the last review. This revised paper is now acceptable.Reviewer #2: (No Response)Reviewer #3: NO further comments. This paper is now acceptable.This study is presented by a group that is leading the world in non-clinical and clinicalresearch on hydrogen gas.**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article (If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.Reviewer #1: NoReviewer #2: NoReviewer #3: No 10 Jun 2020PONE-D-20-11712R1 Pharmacokinetics of a single inhalation of hydrogen gas in pigs Dear Dr. Sano:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! 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In both humans and dogs sleep spindle occurrence between acquisition and recall of a specific memory correlate with learning performance. However, it is not known whether sleep spindle characteristics are also linked to performance beyond the span of a day, except in regard to general mental ability in humans. Such a relationship is likely, as both memory and spindle expression decline with age in both species . We investigated if spindle amplitude, density (spindles/minute) and/or frequency (waves/second) correlate with performance on a short-term memory and a reversal-learning task in old dogs (> 7 years), when measurements of behavior and EEG were on average a month apart. Higher frequencies of fast (≥ 13 Hz) spindles on the frontal and central midline electrodes, and of slow spindles (≤ 13 Hz) on the central midline electrode were linked to worse performance on a reversal-learning task. The present findings suggest a role for spindle frequency as a biomarker of cognitive aging across species: Changes in spindle frequency are associated with dementia risk and onset in humans and declining learning performance in the dog. They are commonly distinguished in a slow  and fast  subtype6.Sleep spindles are brief trains of rhythmic activity, at least half a second in durationCanis familiaris). A shared anthropogenic environment and evolutionary adaptation to its dynamics7 characterize dogs as an animal model for human conditions in general8, and they have specifically been argued a favorable model in comparative neuroscience as well9. Moreover, there is recent evidence11 that, in dogs, transients oscillating in the 9–16 Hz frequency range, corresponding to the broad definition of the sigma band or spindling frequency in humans13, are analogous to human sleep spindles  used the same search criteria. The two studies are therefore directly comparable with each other and the present study. These dog spindle investigations have replicated several findings in the human literature  was recorded at different time points or across age-groups it was only compared with immediate post-sleep behavior32 (the examples include research on early development and IQ). A third common line of research has revealed associations between changes in spindle characteristics and the risk for developing dementia, however the syndrome was not broken down into specific domains (e.g. memory or problem solving decline)34 so that it is not known which functions specifically change as spindle expression changes.Age seems to affect both memory38, and simple exposure to novel information, which increases spindle occurrence in humans41, rats42, and dogs10. It seems, therefore, that without controlling for hormonal levels and pre-sleep experience, comparing spindle activity and memory performance measured further away than a day from each other could be problematic. On the other hand, relatively stable spindle occurrence has been reported within individuals, across nights43, and the frequency ranges of spindling are also assumed to be relatively stable within individuals44. Furthermore, age-related decline of spindle occurrence and amplitude in humans29 are a convincingly replicated effect which is not masked by the expected fluctuation.Day-to-day fluctuations in spindle occurrence and frequency may explain why difference score comparisons have mostly been avoided. Sources of such variation include the menstrual cycle41, but also frequency45 of spindles. Same-day correlates between sleep spindles and learning are thus of low diagnostic value. It has been established that spindle amplitudes and duration in humans correlate with IQ even when each is measured more than a day apart from the other46. No work has investigated the same for learning and memory, and the effect sizes for IQ are also modest, though well-replicated. From a clinical point of view, it is a very important question concerning possible applications of diagnostic EEG in the veterinary praxis if a measurement can predict performance further apart in time.In conclusion, differences in spindle characteristics measured between widely spaced time intervals could be useful biomarkers as they more likely reflect developmental or age-related changes, but more studies need to undertake difference score comparisons to test this assumption. Moreover, although useful for studying memory consolidation, same-day correlates of memory and learning are unlikely to reflect the general performance level of the individual. This is because of the already discussed sources of day-to-day variation in, for instance, occurrence11 to a data-set containing two measurements per dog, of each EEG and behavior  to evaluate whether canine spindles are useful markers of cognitive aging. First, we investigated if changes between two widely spaced (at least 3 months apart) samples of EEG can predict changes in performance between two similarly timed measures of the two learning tasks. Second, because the interval between corresponding probes of EEG and behavior vary from several days to about a month (on average) the data also allowed us to test if any spindle features can function as markers beyond the span of a day. Finally, we focused on a sample of exclusively older dogs (minimum age = 7 years), as variation in performance in elderly individuals is likely better suited to observe markers relevant for distinguishing healthy and pathological aging. The sample from which the present selection of dogs was taken (based on the availability of EEG data) was demonstrated before to display a variability in cognitive performance47.In the present study we apply the same automatic spindle detection algorithm as previously used in the dog48 and dogs11. Importantly, as done in many human studies, we separately analyzed fast and slow spindles11 as in both humans and dogs age-related changes in spindle characteristics differ between the slow and fast variety and topographically .We hypothesized that occurrence, measured as density (spindles/minute), and/or amplitude would show a positive association with performance on the two tasks, while we expected an inverse relationship with spindle frequency, as it rises with age in humanswww.applied-ethology.org) for the use of animals in research. Non-invasive behaviour and EEG tests are not considered as animal experiment and are therefore allowed to be conducted without any special permission from the University Institutional Animal Care and Use Committee (UIACUC). The study was performed in strict accordance with the recommendations in the International Society for Applied Ethology guidelines for the use of animals in research.The behavioural observations conducted in this study complied with national and EU legislation and institutional guidelines and according to Hungarian legislation . The Hungarian “Animal Experiments Scientific and Ethical Committee” approved the experimental procedures under the numbers: PE/EA/2019-5/2017 and PE/EA/853-2/2016. Owners provided written consent to their dogs’ participation. The information included the owner’s right to withdraw their consent at any time. Owners could at any point decline to participate with their dog and could request their data not to be used and/or deleted after collection. The study was performed in accordance with the recommendations in the International Society for Applied Ethology guidelines , 33 females , 49 neutered , 28 from mixed breeds, were included in the current study, with at least one pair of corresponding measurements for each the short-term memory tests and EEG  used to study the effects of ageFrom all EEG recordings available for analysis (two per dog), 18 were excluded for being damaged or not containing sleep. For testing the main effects of spindle amplitude and frequency, recordings were additionally excluded if their signal contained no spindles in spite of sleep . In analyses concerning reversal-learning, subjects were also excluded if they did not attend both reversal-learning measurements (N = 7). Figure legends 3 and 4 in the result section show the final sample size in each analysis.st and 2nd measurements of the short-term memory tests, while the distance between each short term memory and reversal-learning test was 11.6 ± 8.9 days (M ± SD). The distance between the first EEG and first test of short-term memory, as well as 2nd EEG and 2nd memory test, was 31.2 ± 48.2 days (M ± SD), the same distances for EEG and reversal-learning: 28.4 ± 44 (M ± SD). For a schematic overview of the relative average temporal distances between the six measurements, see Fig. EEG measurements, as well as the reversal-learning and short term memory tasks, were performed on different occasions, thus the repeated design required dogs and owners to participate on six testing occasions in total. 137 ± 45.9 days (M ± SD) passed between the 1Three electrodes were placed on the skull midline. The anterior midline electrode (Fz) and central midline electrode (Cz) were referenced against Pz, which was placed on the occipital bone at the back of the dog’s head. The remaining head electrodes consisted of a ground electrode, placed on the left musculus temporalis and two additional electrodes for measuring eye movements (placed on the left and right os zygomioticum). Furthermore, electrocardiogram (ECG), respiration and muscle tone were monitored in order to aid sleep stage identification. The impedance of the active electrodes was kept below 20 kΩ. The signal was collected, pre-filtered, amplified and digitized . The hardware passband was set at 0.5–256 Hz, sampling rate: 512 Hz, anti-aliasing filter cut-off frequency set at 1 kHz, and 12-bit resolution covering a voltage range of ± 2 mV. The second-order software filters  were implemented using System Plus Evolution software .et al.11 and uses criteria proposed by Nonclercq et al.44. Importantly, the method makes use of a two-step procedure and a spectral analysis with a moving, overlapping time-window of 0.5 seconds length (the minimum duration of spindles1). In the first round, the algorithm ‘searches’ for time-windows, within a filtered (between 5–16 Hz) non-REM signal, in which the maximum power is in the sigma range (9–16 Hz)13. The initial detections are used to calculate new search criteria for a second and final round, by taking the maximum likelihood estimates for the mean and standard deviation of each amplitude and frequency and excluding detections in the second search which are outside two standard deviations from the mean. For analyses distinguishing fast and slow spindles, the final count of spindling events is separated into slow and fast spindles using 13 Hz as a cut-off value , that is, detections with frequency ≤ 13 Hz is categorized as slow, ≥13 Hz – fast. The frequency of slow and fast spindles overlaps at 13 Hz . The mean amplitude of a recording was calculated as the average across spindle detections in that recording of the root-mean-square value for the signal segments corresponding to spindling events.The method used to detect spindles is described in Iotchev 23. Briefly, five equally shaped, open containers were positioned in equal distance from each other in a semi-circular arrangement, resulting in each container being two meters away from the starting position of the dog . The task was repeated five times and each container was baited once in a random order. The measured variables were number of initially correct responses and number of wrong choices (since dogs had the possibility to visit any number of containers until they found the right location). However, in accordance with the conclusion of the original study23 we only use the number of initially correct choices in below analyses.For the detailed protocol please see49. Briefly, dogs were first trained to either associate i) the spatial location (left or right) or ii) physical characteristics  of an object with the presence or absence of food . The assignment to either reversal learning condition was counterbalanced between dogs and the conditions were switched between test and re-test to avoid carry-over effects.For the detailed protocol please see49; therefore, we expected younger dogs to perform better than old dogs in both tasks.In the spatial location condition the task relied on egocentric spatial coding , specifically relying on learning  and reversal-learning (executive function). For the physical characteristics condition, the tasks relied on visual learning and reversal learning (executive function). Both tasks also relied on visual discrimination-learning and reward and object approach-learning (learning domain). Previous findings suggested similar effects of age on location and size discriminationet al.47 and follows criteria based on Kis et al.50. The paradigm was used to study discrimination learning before reversal-learning was tested. Importantly, dogs were included in a reversal-learning task only if they passed a threshold for learning the initial association . The reversal-learning consisted of a maximum of 50 trials during which the rules were reversed . The present study makes use of the variable trial to criterion, that is, how many of the 50 trials were required for the dog to reach the threshold described for the preceding discrimination learning task. The reversal-learning part of the task was chosen instead of the discrimination learning part, due to its association with inhibition (as in suppression) as an underlying mechanism53, since it is hypothesized that spindles also support memory consolidation by suppressing irrelevant information54.Dogs were presented with the positive and negative stimuli in consecutive trials; the stimuli were presented in a pre-determined pseudo-random order, with no more than two trials of the same type being presented consecutively. The experimenter put the plate on the floor and as soon as the plate was on the floor, the owner unleashed the dog. Owners were instructed to unleash the dogs exactly as the plate touched the floor, so to avoid biasing the dog’s behavior. The procedure is described in detail in Piotti The structure of the data allowed us to investigate two questions. Firstly, whether a difference between the first and second recording of EEG is related to changes in performance between the first and second measure of short-term memory task and reversal-learning. Secondly, whether corresponding spindle and behavioral measures correlate when measured further apart than a day.38. The need to remove outliers was based on the standard scores for spindle amplitude. Subjects with values above or below 2.68 standard deviations from the group mean were excluded from the analyses . The 2.68 threshold is derived from the quartile-based rule for detecting outliers, applied to the value likelihoods expected in a normal distribution (see for example http://www.cs.uni.edu/~campbell/stat/normfact.html). Based on previous observations in the dog11 and the human literature3, misleading extreme values are most likely to occur for spindle amplitudes.All analyses shared the same basic structure. Correlation matrices (Pearson) were calculated involving one of three behavioral tasks (the short-term memory task and each (starting) condition of the reversal-learning task were never tested at the same time for reasons explained below), three spindle-derived measures  and age (in years) at the beginning of the experiment (prior to the first measurement). All analyses were conducted independently for both active electrodes  and spindle type . We did not include sex in analyses on the present sample, because more than 80% of the animals were neutered and sex differences in sleep spindles depend on sexual hormones in both humans and dogsnd – 1st measurement) for each spindle measure and the performance on the short-term memory and reversal-learning tasks. Therefore, we had 6 difference scores (DS):(1) three EEG-derived DS for each density, amplitude and frequency of spindles, (2) short-term memory DS, (3) spatial location DS, (4) physical characteristics DS.Towards answering the first question, whether spindle measures and learning performance changed together, we calculated difference scores . Overall, analyses based on difference scores consisted for each electrode and spindle type of three correlation matrices – one across all dogs including only the difference scores for the short-term memory task as a behavioral measure and one for each starting condition on the reversal-learning task and including only difference scores on the reversal-learning task as a behavioral measure. In total 12 analyses tests) based on difference scores were performed.Separate correlation matrices were calculated for whether difference scores on the short-term memory task or trials to criterion on the reversal-learning task were included. For analyses based on the reversal-learning task we had to separately analyze each starting condition in accordance with earlier findings suggesting a strong effect of condition on the animals’ performanceAnalyses based on raw  scores were controlled for the distance between corresponding behavioral and EEG measurements by using distance (in days) as a control variable in partial correlations.47 would preclude the grouping of animals within a series of measurements, but instead it would be of interest how each condition affects spindle-performance associations across recordings obtained under the condition of interest. These resulted in four analyses based on raw scores per electrode and spindle type and 16 analyses ((2 electrodes × 2 spindle types) × 2 conditions  and/or × 2 measurements (for short-term memory)) in total.For each electrode and spindle type, each series of recordings (first behavior and first EEG or second behavior and second EEG) were tested separately including only performance on the short-term memory task as a behavioral measure. Analysis for performance on the reversal-learning task, instead looked at each condition separately, but across all recordings (first and second behavior and EEG) obtained with the condition. We reasoned that the effect of condition observed previouslyDue to the overlap in variables included across correlation matrices, several comparisons were repeated under different sub-sets of the data. These concern correlations between spindle-derived measures and correlations with age. Due to this redundancy, we only consider correlations between spindle-measures and with age, from the largest sub-samples: 4 correlation matrices  including performance on the short-term memory task, which were not separated by condition as those concerning the reversal-learning task.st or 2nd) × 2 spindle types), resulting in a Bonferroni significance threshold of 0.00625. Independence, which is required for Bonferroni55 was defined here either as samples not overlapping in subjects ; samples not overlapping in recordings (recordings from different measurements i.e. 1st or 2nd); or non-overlapping populations of spindles (fast versus slow). Below we report and plot findings which were significant at least prior correction and associated with performance on the learning and memory tasks for better overview. In a tabular overview  resulting in 8 independent multiple comparisons which we corrected using Bonferroni. In general, groups of tests to which Bonferroni was applied consisted of 8 comparisons (2 electrodes × 2 conditions/measurements (1 ×2 ×2 rehttps://www.psychometrica.de/correlation.html) employing a z-test method described in Eid, Gollwitzer and Schmidt (2011)56.When similar correlations were tested twice (for different (starting) conditions of the reversal learning task or for the first and second series of measurements) and yielded different results we compared the correlation coefficients with an online calculator . Figures were made with GraphPad Prism.In the reversal-learning test, for animals which started in the physical characteristics condition there was a negative correlation between the difference scores for trials needed to criterion and fast spindle frequency on Fz . This correlation was not significantly different from the same test for the first series of measurements  which was not significant . This correlation was not significantly different from the same test for the first series of measurements  which was not significant . This correlation was not significantly different from the non-significant correlation obtained for the first series of measurements  . This correlation was not significantly different from the one obtained for the first series of measurements  which was not significant  thus this sample might also simply have been underpowered for the question at hand.Increases in fast spindle frequency over Fz and Cz, as well as slow spindle frequency over Cz correlated with worse performance on the reversal-learning task for dogs which began in the physical characteristics condition. Across recordings obtained in the spatial location condition on Fz the raw scores for fast spindle frequency and trials needed to criterion were also positively correlated. As a note of caution, however, only half of the correlations between spindle frequency and reversal learning remained significant under a Bonferroni correction Table . A previ48 and are not always replicated4. However, Ktonas and colleagues, showed that dementia patients  displayed faster instantaneous spindle frequencies as compared to healthy controls33, which together with the here described findings makes spindling frequency the first marker of cognitive aging to be observed across species. Concerning possible mechanisms, two alternative scenarios could explain an age-related increase in spindling frequency as well as the here observed association of higher frequency with worse performance. By analogy, we could assume that similar to what is seen in the dopaminergic system, increased frequency is a compensatory response to cell-loss, exhibited by the surviving cells57. Alternatively, because the degree of suppression in the cortex can alter spindling frequency58, with the lowest frequencies exhibited during deep anesthesia, it can be reasoned that higher spindling frequencies reflect loss of cortical inhibition, which characterizes the aging process in humans59. Inhibition is also a crucial element in reversal-learning, via suppression of the original associations which ought to be reversed53. In favor of the former interpretation, we observed that on Cz, where increases in slow spindle frequency were correlated with worse reversal-learning performance, slow spindle density and frequency, as well as their difference scores, were also negatively correlated across several selections of data (Supplementary). However, fast spindle frequency and density (raw and difference scores) were instead positively correlated on both Fz and Cz. This effect also remained significant across different data-selections. It is not clear, yet, what the latter finding could tell about the underlying causal dynamics, but fast spindle density was previously found to increase with age in dogs11 and seems unique to the canine aging process. Since dogs, like humans, show a predominantly frontal decline in spindle amplitude26, it is possible that in dogs both frequency and density of fast spindles are augmented as a compensatory response.These results are somewhat surprising, as in humans age-related increases in frequency are not a frequent observation10 and human62, were associated with performance on either of the two tests, when behavior and EEG had been measured with distances greater than a day. Indeed, density is a likely unstable measure of spindle activity due to the influence of hormonal changes37 and diurnal experience42 on spindle occurrence, although in the present sample, of mostly neutered animals, only the latter applies. The link between spindle occurrence and post-sleep improvement on novel tasks is generally controversial. Although mechanistic work in animal models suggests a plausible explanation for such observations63 the largest (N > 900) study in humans to date could not confirm this notion20, moreover different factors appear to play a role in whether such an association is observed. These include timing relative to ripples and slow oscillation up-states19, as well as schema-compliance of the material to be encoded17. Amplitude was repeatedly found to correlate with at least IQ, but the strength of the association is modest46.Neither density (spindles/minute) nor amplitude, which if measured between acquisition and recall correlate with memory performance in the dog64, long time intervals could render allegedly corresponding EEG and behavioral measurements incomparable due to developmental changes. The interval between the two behavioral tests and EEG was an important control factor in our raw score analyses, because it was not fixed and varied from subject to subject. We therefore performed partial correlations controlling for distance (in days) between corresponding measurements of behavior and EEG.Important limitations of the present study are the high variation in time elapsed between corresponding measures of memory and EEG (for example 31.2 ± 48.2 days (M ± SD) for EEG versus short-term memory measurements). We can assume that due to developmental and age-related changes in spindle activity, confirmed also in the dogA higher incidence of complete fast spindle absence in dogs was another problem. The overall data-loss for fast spindle analyses was between 29 and 30 dogs on Fz and Cz, respectively .65. In support of this assumption, in the present study age and performance on the short-term memory task were associated only in the second series of measurements. As the sample of dogs for which EEG data was available is a sub-sample of the study in which an aging effect on the short-term memory task was originally described23 this asymmetry suggests that the 3 month interval between measurements captures a long-enough segment to observe age-related cognitive decline. At the end of the chosen interval the effect was strong enough to be observed also in the here reported sub-sample. Alternatively, sub-samples in which age with short-term memory were not correlated present Type II errors. The significant and non-significant correlations between memory and age were not significantly different from each other and the larger sample used in Piotti et al. suggests that performance on this task does decline with age23.The distance between first and second measurements on each behavior and EEG was always at least 3 months. We deemed 3 months a considerable interval in dogs, because, for example, in many breeds reaching sexual maturity takes less than a year53, was associated with spindle characteristics in this sample. In the human literature, IQ is also associated with spindle characteristics independent of the distance between measurements of EEG and behavior46. Most measures of memory in humans and dogs, on the other hand, have been demonstrated to correlate with spindle occurrence  only when spindles have been obtained from between acquisition and recall on the same day62. However, IQ-spindle associations concern amplitude and duration, and though well-replicated the effect sizes are small46.It is worth noticing that only reversal-learning, recognized in the literature as a fluid cognitive operation11, no distinction had been made between the cognitive performance of individuals, whereas the aged dogs in the present sample were compared on two tasks of memory and learning. A pattern that emerges in comparison between both studies is that high fast spindle frequency in dogs (here on both Fz and Cz) is characteristic of both aging and performance decline, but this notion will require more empirical support. Currently the only similar study in humans relies on a fairly small sample33, while this first finding in the dog should be replicated in a more homogenous sample, with for example only one condition for reversal learning.Although a previous study suggests that age-related frequency increase in dogs is more pronounced over CzSupplementary.Supplementary Dataset 1.Supplementary Dataset 2.Supplementary Dataset 3.


The extension of the interdialytic interval due to due to dialysis session non-attendance varies according to which session of the week the patient misses. The impact of this on subsequent hospitalization and mortality is unknown.The ARO cohort study prospectively collected data from hemodialysis patients across 15 European countries on demography, comorbidity, laboratory, hospitalisation, mortality and individual hemodialysis sessions from 2007 to 2014. Event rates for death and hospitalisation according to dialysis day of the week were calculated for patients who attended the three previous scheduled hemodialysis sessions, who then on the next scheduled dialysis day either attended or did not attend. The hazard ratio for these events following non-attendance for the first compared to the second dialysis session of the week was estimated using Cox proportional hazards model adjusted for patient demographics.3.8 million hemodialysis sessions in 9397 patients were analysed. The non-attendance rates for Monday/Wednesday/Friday sessions were 0.8, 0.9% & 1.4% respectively, and for Tuesday/Thursday/Saturday sessions were 0.6, 1.0% & 1.2% respectively. Compared to those who attended, for the 48–72 h between non-attendance and the next scheduled haemodialysis session, mortality significantly increased from 4.86 to 51.9/100 pt-yrs and hospitalisation increased from 0.58 to 2.1/yr. As time from the two-day break increased, the risk associated with non-attendance lessened: compared to missing the second hemodialysis session, missing the first session had a hazard ratio for mortality of 2.04 (95% CI 1.27–3.29), and for hospitalisation 1.78 (95% CI 1.29–2.47). In patients who attended their scheduled dialysis session and the three preceding, after the two-day break there were absolute increases in mortality (8.3 vs. 4.9/100 pt-yrs) and hospitalisation (1.0 vs. 0.6/yr for the rest of the week) comparable to previous studies.In addition to hospitalisation and mortality increases seen after the two-day break, additional harm may be manifested in the greater increases in mortality and hospitalisation observed after non-attendance for the first hemodialysis session after the two-day break compared to missing other sessions. Non-attendance for dialysis is common, with the proportion of sessions affected between 0.6% in Europe and Japan to 7.9% in the United States . Non-adhOver 95 % of patients worldwide receiving (HD) for treatment of kidney failure are offered three dialysis sessions per week, with the most commonly assigned schedules being Monday/Wednesday/Friday (MWF) and Tuesday/Thursday/Saturday (TTS) . The HD This study explored the hypothesis that there may be differential risk of mortality and hospitalisation following non-attendance according to the interdialytic interval.The Analyzing Data, Recognizing Excellence and Optimizing Outcomes (ARO) cohort study contains anonymised longitudinal individual-level data for patients starting HD who were enrolled at one of the 312 Fresenius Medical Care (FMC) facilities across 15 European countries between 2007 and 2009 and followed up until the end of 2014. Data on demography, comorbidity, laboratory, hospitalisation, mortality and individual HD sessions were captured prospectively [In patients who were prescribed three times a week HD, for each day of follow-up time we reviewed the preceding seven days to identify on which days HD was conducted, enabling us to identify MWF and TTS schedules. We did not analyse patients receiving the less common Tuesday/Thursday/Sunday schedule, as TTS patients could miss a Saturday session, and be offered a Sunday session instead. This would be interpreted as a Tuesday/Thursday/Sunday schedule and would artificially lower the rate of non-attendance on Saturday in TTS patients. The first HD session after a scheduled long interval was defined as the first day of the dialysis week, this being Monday for patients dialysing MWF and Tuesday for patients dialysing TTS.We examined each scheduled dialysis day, first confirming that the three preceding scheduled MWF or TTS sessions had been attended. We then assessed for non-attendance determined by the absence of a recorded pre-dialysis blood-pressure, pre-dialysis weight and dialysis treatment time. From these we excluded dialysis days and associated events in patients who were hospitalized or died on the identified day of non-attendance as these events which prevent attendance would not represent non-adherence. We also excluded patients who had been discharged from hospital in the preceding seven days and those who had an un-scheduled HD session on the preceding day potentially resulting in a temporary or permanent change to their dialysis schedule. Patients who had missed an HD session but then return to a MWF or TTS pattern then re-entered the data set from this point. A record of hospital admission is made by the clinic staff and those are required when a patient missed at least one treatment. The analysis was limited to hospitalizations lasting longer than one night, which do not involve routine dialysis vascular access care .Although all follow-up time on 3xW HD was considered in our analysis, in order to facilitate comparisons to other studies of non-adherence, the demographics of the cohort are reported stratified by whether the patient did not attend a HD session during the first 4 months of the study. We report the crude non-attendance as a proportion, the numerator being the number of missed scheduled HD sessions in non-hospitalised patients, who had an established MWF or TTS schedule and who have attended all three preceding scheduled HD sessions. The denominator was the number of scheduled HD sessions in the above patients and setting. These crude proportions are reported across dialysis days and between schedules, using multivariate logistic regression to determine if significant variation existed.In order to quantify the per-day rates of hospitalisation and mortality and their relationship to the dialysis schedule we calculated hospitalisation (per patient year) and mortality (per 100 patient years) event rates for each individual dialysis day in individuals who attended the three preceding scheduled dialysis. These events are reported separately for the two dialysis schedules day and if the patient attended or did not attend the next scheduled dialysis session. For patients classified as not attending this HD session we reported hospitalisation and mortality rates for each day between the day after the missed session and the day of the next scheduled HD session (2 to 3 days), as shown in Fig. The proportion of sessions fully attended prior to hospitalisation was estimated to determine the proportion of patients deviating from their schedule prior to admission: the numerator being the number of hospitalisations in which patients deviated from their scheduled HD sessions prior to hospitalisation, the denominator being all hospitalisations in three times a week patients on established MWF or TTS schedules.We conducted the following sensitivity analyses. To explore if rescheduled or emergency dialysis identified through an FMC provider altered the variation in outcomes according to dialysis day of the week, we censored at the time when rescheduled dialysis was delivered. In an attempt to identify facility-directed dialysis rescheduling (e.g. moving from MWF to TTS) as an explanation for variation in non-attendance, we excluded non-attended sessions which preceded a new three times a week schedule that persisted for two weeks. All analyses were conducted in R version 3.4.4.From the 11,211 patients in the ARO cohort, 9397 were identified as receiving three times a week HD, could be assigned a MWF or TTS schedule, had complete set of covariate data and had survived 90 days from entry into the study. The overall mortality rate was 11.0 deaths per 100 patient years, and the emergency hospitalisation rate was 0.66 per patient year. Figure Following three consecutive attended HD sessions, the proportion of scheduled HD sessions that were not attended was 1.0% . The characteristics of patients who attended compared to those did not attend one or more sessions during the first four months of the study are detailed in Table P = 0.32).A higher proportion of the third HD session were missed in both MWF and TTS schedules Table . CompareOverall, between a missed HD session and the next scheduled HD session, the mortality rate was 51.9 per 100 patient years 95% CI 43.5–61.4), and hospitalisation rate was 2.13 per patient years (95% CI 1.95–2.32), compared to 4.86 (95% CI 4.6–5.1) and 0.58 (95% CI 0.57–0.59) during the same period in those who attended the HD session. The variation in hospitalisation and mortality events and associated event rates, stratified by attendance or non-attendance across the dialysis week is reported in Table .5–61.4, In patients who attended their scheduled dialysis session and the three preceding HD sessions, mortality was greatest on the day of the first HD session after the long interdialytic interval in both MWF and TTS schedules Figs.  and 4. MOur sensitivity analysis which censored on the day a rearranged dialysis session was performed prior to the next scheduled dialysis did not show a significant difference in the hazard of mortality following non-attendance according to which day of the dialysis week was not attended, however hospitalisation in TTS patients after non-attendance for the third session did increase (Figure S2 in Additional File There is mounting evidence of the size and mechanism of harm from the two day break in three times a week dialysis. We were able to use session-level data from 9397 patients spanning fifteen countries to show that this harm expands to the period immediately after non-attendance with greater increases in hospitalization and mortality after the long interdialytic interval. Non-attendance was greatest for sessions immediately prior to or scheduled for a weekend day, and even after prior full HD attendance three times a week, there are increases in mortality and hospitalisation endpoints after the long interdialytic interval.Due to the variation in definitions for adherence in the published literature, the assessment of non-adherence being performed on the period immediately prior to the patient consenting into observational studies introducing a range of biases , and theIn limiting our analysis to patients with full attendance, we demonstrate how attendance alters mortality and hospitalisation rates. USRDS and the UK Renal Registry data have shown hospitalisation and mortality event rates approximately twice those observed in our study on the first and second HD days , 10. TheWe cannot determine from these observational data with certainty that missed sessions contribute causally to hospitalisation and dialysis. The relationship could be explained by a confounding factor, such as acute illness which both prevents attendance for scheduled dialysis and leads to hospital admission or death, or that the true timing of death was before the non-attended session. However, it is difficult to explain how these mechanisms which could bias our findings would vary across the dialysis week, since the prevalence or severity of acute illness itself is unlikely to vary to the degree required to explain our observations. This variation could plausibly represent a manifestation of ongoing harm caused by the two-day break manifest beyond the first session of the dialysis week. Our study does not attempt to shed additional light on the likely mechanisms of harm associated with the two-day break, but it does support the conclusion that the long gap is followed by increases in mortality and hospitalisation.Our study has several notable strengths. The most important being that through utilisation of session level data, we were able to confidently assign and ensure that patients received the scheduled three times a week dialysis for the preceding week in both non-adherent and fully attended analyses. Our analysis makes it unlikely that variation in non-attendance across the dialysis week, with greater non-attendance before the two day break, explains increases in events after the long interdialytic interval. We may also have provided a more accurate measure of the increased risk associated with the long gap than previous studies. Our study is limited by the fact that we have adopted a definition of non-attendance based on the available data, although our rates are similar to those reported by others . To imprFrom our study, we suggest how a clinician should consider the potential implications of a patient not attending a haemodialysis session should account for which session is missed. Our data also indicate that even following efforts to reschedule dialysis in the 24–48 h after non-attendance, a greater hazard for mortality and hospitalization persist after non-attendance for the HD session after the long interdialytic interval. In fact, the extension of the interdialytic interval from one to two days, even in in patients who attend regularly, is associated with poor outcomes. We hope that our data will help physicians and nurses counselling patients regarding the potential harms of missed dialysis sessions. We believe that with accumulating observational data suggesting clinical harm associated with the long interdialytic interval observational analysis of practices excluding it should inform the design of a clinical trial of dialysis regimens with and without a two-day gap.Additional file 1. Country specific non-attendance rates according to session of the dialysis week; Comparison of hazard associated with non-compliance according to dialysis day of the week based on primary analysis, with established changes in dialysis schedules excluded, and censoring at first rearranged dialysis session; statistical justification for summing across days. Figures showing sensitivity analyses outline in main text, methods.


Despite the potentially protective effects of younger demographics, the closer intergenerational contact, limitations on health care facilities, and frequency of comorbidities in lower-income countries require sustained nonpharmaceutical interventions (NPIs) to avoid overwhelming health care capacity. As a result of strict NPIs, the protective effects of immunity will be reduced, and it will be important to improve testing capacity. Ensuring equitable provision of oxygen and—when they are ready—pharmaceutical interventions should be a global priority.Lower-income countries have recognized the potential impact of coronavirus disease 2019 (COVID-19) from observing ongoing epidemics. Many have intervened quickly and early with measures to slow viral transmission, which may partly explain the low rates observed so far in these countries. Walker Science, this issue p. 413; see also p. 368 Modeling reveals differences in the unfolding COVID-19 epidemics and responses to their control among countries with different income levels. The ongoing coronavirus disease 2019 (COVID-19) pandemic poses a severe threat to public health worldwide. We combine data on demography, contact patterns, disease severity, and health care capacity and quality to understand its impact and inform strategies for its control. Younger populations in lower-income countries may reduce overall risk, but limited health system capacity coupled with closer intergenerational contact largely negates this benefit. Mitigation strategies that slow but do not interrupt transmission will still lead to COVID-19 epidemics rapidly overwhelming health systems, with substantial excess deaths in lower-income countries resulting from the poorer health care available. Of countries that have undertaken suppression to date, lower-income countries have acted earlier. However, this will need to be maintained or triggered more frequently in these settings to keep below available health capacity, with associated detrimental consequences for the wider health, well-being, and economies of these countries. The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus is a major global health threat, with 5.4 million cases and 344,000 deaths confirmed worldwide as of 26 May 2020 (R0) of 3.0 (a 3.5-day doubling time) and investigate scenarios with R0 between 2.3 (a 5-day doubling time) and 3.5 (a 3-day doubling time).We collated data on global demographic projections of population size by age and country and available data on social mixing patterns by age and country-level income category. We first include these within a simple SIR modeling framework –based approach to model the likely availability of hospital beds  on the basis of a suite of relevant health care–related and socioeconomic covariates . This prediction of hospital bed capacity was then combined with estimates of intensive care unit (ICU) beds  across a range of different settings  identified through a systematic literature review . These eThe boosted regression tree model predicts hospital bed capacity well across the range of countries for which data were available . We findTo understand the potential impact of weaker health systems on the IFR, we collated expert clinical opinion on the likely outcomes for COVID-19 patients . These expert estimates drew on recent experience treating COVID-19 patients in the United Kingdom (UK) alongside an understanding of severe pneumonia outcomes in LIC and LMIC settings. Mechanical ventilation (MV), which has been required by 80% of COVID-19 ICU patients in the UK , and 60%There remain large uncertainties in the underlying determinants of the severity of SARS-CoV-2 infection and how these translate across settings. However, clear risk factors include age . Using GIn LICs and LMICs, there is a higher burden of infectious diseases such as HIV/AIDS and tuberculosis (TB), and of poverty-related determinants of poorer health outcomes such as malnutrition, than in HICs. These generally occur in younger populations . AlthougRt remains above 1 and hence a single-peaked epidemic is predicted owing to the buildup of herd immunity; and (ii) suppression, whereby transmission is reduced such that Rt < 1 and hence, if interventions are later released, transmission will be expected to rise as herd immunity will not have been achieved.To understand the consequences of the demographic, social contact, and health system patterns on strategies for reducing the spread of SARS-CoV-2 in the context of control measures already enacted by countries, we developed an age-structured SEIR modeling framework to explore the dynamics of the epidemic under different health system capacity constraints . As for the SIR final size calculations, we use demographic data and social contact patterns based on representative settings in the LIC, LMIC, UMIC, and HIC strata. Disease progression for individuals requiring hospitalization was explicitly modeled to track requirements for hospital bed provision for severe pneumonia (those for whom we assume oxygen support will be required) and for ICU provision [those for whom we assume 80% would require MV in line with U.K. data . If “optimal” mitigation based on enhanced social distancing is pursued, for an R0 of 3.0, we estimate a maximum reduction in infections in individual countries in the range 30 to 38% (median 33%) and a range of reduction in mortality between 19 and 55% (median 39%) . These optimal reductions in transmission and burden were achieved with a range of reductions in the overall rate of social contact across countries between 40.0 and 44.9% (median 43.9%), with this range across countries increasing to 42.9 to 47.9% (median 46.9%) for an R0 of 3.5 and decreasing to 31.4 to 35.8% across countries (median 35.0%) for an R0 of 2.3. Combining mitigation with enhanced social distancing of elderly individuals is predicted to result in greater mortality reductions of 23 to 67% across countries (median 49%) for R0 = 3 (table S5). However, both of these strategies are predicted to have a lower proportional impact in lower-income settings compared to higher-income settings: median reductions in mortality in the range of 19.5 to 41.6% (median 25.3%) in LICs, in contrast to 21.5 to 55.1% (median 49.9%) in HICs for optimized mitigation strategies including social distancing, and in the range of 25.4 to 50.9% (median 32.6%) in LICs in contrast to 23.4 to 66.6% (median 60.1%) in HICs for optimized mitigation strategies including enhanced social distancing for elderly individuals (table S5). This lower proportional reduction in deaths of mitigation scenarios in lower-income settings is driven by the more homogeneous contact patterns by age in these settings  (below 1)  and B. HThe comparative benefits and drawbacks of these scenarios  will differ between settings depending on their health care capacity and quality. In all settings, although our optimized mitigation scenario is predicted to substantially reduce the gap between demand for hospital beds and capacity, demand for critical care is still predicted to vastly exceed capacity, leading to a substantial additional burden relative to a scenario with unlimited capacity . AlthougWe estimate that constraints on health care capacity would be likely to increase the IFR under a mitigation strategy in all settings. However, as has been observed, HICs and UMICs are likely to be able to put in place surge capacity to limit any impact on mortality  and LMICs, a country-level median of 19.3 . By contrast, HICs reported a country-level median of 72.6  cases per reported death. The extent to which reported case-to-death ratios are a reliable indicator of relative case or infection (including asymptomatic) ascertainment rates will depend upon trends in IFR, the reproduction number R0, and the extent to which deaths are reported, all of which are liable to vary across income strata. However, this trend is suggestive of the extent to which testing capacity will need to be developed in LIC and LMIC settings if approaches such as case identification coupled with contact tracing are to form part of a successful mitigation or exit strategy.We used European Centre for Disease Control (ECDC) data prior to the date of implementation of suppression or, in the absence of identified suppression measures, the date of last entry within the ACAPS dataset. We then evaluated the ratio of reported cases to deaths in this period to provide a measure of the capacity of countries to contain transmission through testing-based approaches prior to, or in the absence of, suppression. Our estimates show clear differences by region and gradient across income strata: LICs with three or more deaths prior to suppression reported a country-level median of 8.6 cases per reported death  for the implementation of transmission reductions and modeled these as lasting for 30 days before being lifted . Reimplementation then occurs if the trigger threshold is eclipsed again. The level of reduction in contact rates will determine the speed at which the infected population is depleted during the intervention  will be achieved for lower trigger levels; however, this is balanced by a slower buildup of herd immunity such that the interventions would need to remain in place for longer in the absence of a vaccine . For each scenario, we modeled the first suppression to have been initiated at the median threshold for the setting that has been observed to date (i.e., LICs and LMICs initiate earlier than UMICs and HICs, If ICU demand is to be kept below the estimated median ICU capacity for each income strata, all countries are predicted to need to spend a substantial proportion of time in suppression . In all Rt that remain close to R0.Our estimates of increasing time between suppression triggers in HICs in However, these results do not account for other interventions that could be implemented during periods in which suppressive measures are not in place. Once the number of new infections drops to a manageable level, it is likely that more widespread testing and isolation of cases coupled with contact tracing can help to prevent a resurgence of transmission, as has been observed in countries such as South Korea (The results presented here illustrate the potential impact of the COVID-19 pandemic in LICs and LMICs compared to the epidemics that have occurred to date in UMICs and HICs. Our analyses give insight into how differences in demography, social structure, and health care availability and quality combine and potentially influence the impact of measures that can help reduce the spread of the virus. At the current time, it is not possible to predict with any certainty the exact number of cases for any given country, the precise mortality and disease burden that will result, or the benefits and drawbacks of the different approaches to controlling the virus that are currently being implemented. A full understanding of these will only be available retrospectively.Although our results illustrate the challenges that many countries will face in attempting to mitigate the impact of local COVID-19 epidemics, it is important to bear in mind that even moderate levels of changes in behavior can avert many infections and hence save millions of lives (Rt below 1 (It is also important to note that we do not quantify the wider societal and economic impact of the intensive mitigation or suppression approaches; nor do we address the challenge of intensive suppression initiatives in LICs and LIMCs, where a high degree of informal labor makes such interventions challenging and may limit the extent to which they can reduce Our results highlight the difficult decisions that countries are faced with in the coming weeks and months irrespective of region or income status. Given the likely worse prognosis of severe COVID-19 cases in settings with weaker health systems, coupled with the higher vulnerability of developing economies to the negative effects of stringent NPIs, the trade-offs faced by lower-income countries are complex considering the ongoing uncertainty over the most appropriate and effective exit strategies. In the interim, the priority should be to increase the availability of oxygen support to mitigate the health impact alongside enhancing the capacity for surveillance and wide-scale testing to reduce the spread of infection and tailor appropriate NPIs. In the longer term, ensuring equitable provision of pharmaceutical interventions to lower-income countries once they are developed should be a global priority.Our analysis demonstrates the extent to which countries have mobilized to combat the COVID-19 pandemic. Many lower-income countries have acted while transmission remains at low levels which is likely to have substantially slowed the spread of the virus. In the absence of a vaccine, all governments are likely to face challenging decisions around intervention strategies for the foreseeable future. However, the still relevant counterfactual of a largely unmitigated pandemic clearly demonstrates the extent to which rapid, decisive, and collective action remains critical to save lives globally.