A global correlation between variables was also performed using a two‐tailed Pearson's correlation test. AST rats displayed a higher functional capacity and a lower cardiac remodelling and dysfunction when compared to AS, as well as the myocardial capillary rarefaction was prevented. Regarding metabolic properties, immunoblotting and enzymatic assay raised beneficial effects of exercise on fatty acid transport and oxidation pathways. The correlation assessment indicated a positive correlation between variables of angiogenesis and FA utilisation, as well as between metabolism and echocardiographic parameters. In conclusion, early exercise improves exercise tolerance and attenuates cardiac structural and functional remodelling. In parallel, exercise attenuated myocardial capillary and lipid metabolism derangement in rats with aortic stenosis‐induced heart failure.We employed an early training exercise program, immediately after recovery from surgery, and before severe cardiac hypertrophy, to study the underlying mechanism involved with the amelioration of cardiac dysfunction in aortic stenosis (AS) rats. As ET induces angiogenesis and oxygen support, we aimed to verify the effect of exercise on myocardial lipid metabolism disturbance. Wistar rats were divided into Sham, trained Sham (ShamT), AS and trained AS (AST). The exercise consisted of 5‐week sessions of treadmill running for 16 weeks. Statistical analysis was conducted by  Rats were housed in collective polypropylene cages in a climate‐controlled environment at 23°C with a reverse 12:12‐h light/dark cycle and free access to food and water. All experiments and procedures were performed according to the Brazilian Guide for the Care and Use of Laboratory Animals and approved by Botucatu Medical School Animal Research Ethics Committee (protocol number 1239/2017).Twenty‐one‐day‐old male 2.2Supra‐valvar AS was surgically induced as described previously.n = 16 and AS, n = 16) or submitted to exercise training  for 16 weeks. The mortality rate did not differ between AS and AST. Two AS rats were removed from the study due to an unsatisfactory remodelling related to the clip placement in the aorta. Regarding the AST group, one rat had collateral circulation and two others were unable to adapt properly to the treadmill and were also excluded from the study.Initially, rats underwent either supra‐valvar aortic stenosis (AS) or Sham surgery (Sham). After 2 weeks, the animals were redistributed to be kept untrained , and subsequently, rats were submitted to euthanasia for additional analyses Figure .2.3Functional capacity was assessed before and after the ET period. TET was performed on a motorized treadmill for rats (AVS Projetos). Rats underwent 5 m/min/day test environment adaptation for 1 week. Maximum running speed and total time were recorded. Briefly, TET began at 6 m/min and increased by 3 m/min every 3 min until exhaustion. Exhaustion was defined as the non‐maintenance of the race at the proposed speed. The TET results were also used to prescribe the exercise program.2.4The ET protocol was modified from previous studies.2.5Measurements were performed after anaesthesia by intramuscular injection of a mixture of ketamine (50 mg/km) and xylazine (1 mg/kg), using an apparatus  equipped with a 5–11.5 MHz multifrequency probe, as recently described in detail.2.6The nutritional profile was evaluated according to body weight, fat deposits and food intake; body weight and food intake were measured weekly.post‐mortem  in the AS and AST groups. Two blind observers carried out a subjective evaluation of HF signals throughout the experiment as previously performed.At euthanasia, was observed the occurrence of HF features in vivo (tachypnoea) and 2.7Slices of LV were kept in buffered formalin for 12 h at room temperature, followed by sequential dehydration in increasing concentrations of alcohol, cleared in xylene and embedded in paraffin. The blocks were cut into 3‐μm sections for immunohistochemical staining.For immunohistochemistry, marked and pre‐assembled blades were dewaxed in xylene and rehydrated in decreasing concentrations of alcohol until water. For antigen recovery, the slides were heated at 98°C with citrate buffer (pH 6.0) for 30 min and then incubated in 0.3% hydrogen peroxide at room temperature for endogenous tissue peroxidase block. Then, they were washed with Tris‐buffered saline/Tween 20 (TBST), pH 7.5 and incubated with primary antibodies against VEGF , VEGF receptor 1  and 2  and differentiation cluster 34  for 1 h in a humid chamber at room temperature. Slides were washed again with TBST, incubated with a secondary peroxidase horseradish polymer‐conjugated antibody for 30 min at room temperature, washed with TBST, incubated with 3,3′ diaminobenzidine stain for 5 min and counterstained with haematoxylin. Stereological point‐counting procedures were applied to perform a morphometric analysis.2.8We analysed the expression of cardiac lipid metabolism proteins by the Western blot method as previously described in detail.2.9g for 10 min at 4°C and the supernatant was collected. All enzyme activities were determined at 25°C using a Spectra Max 250 microplate spectrophotometer (Molecular Devices), and the assay buffer without the sample was used as blank. CK was assayed in a medium consisting of 91.1 mM Tris–HCL, 1 mM ATP, 0.262 mM ß‐NAD reduced form, 0.728 mM PEP, 10 mM creatine, 5 mM KCl, 2 mM MgSO4, 10 units lactic dehydrogenase, 0.4–0.6 unit pyruvate kinase and 0.05% Triton X‐100. pH 7.4. The total assay volume was 165 and 2 microliter of homogenate was added. The assay was initiated by the addition of creatine. The absorbance of CS and CK assays was monitored at 412 nm. For OHADH, the reaction contained 100 mM PBS pH 7.3, 0.45 mM NADH and 1.0 mM acetoacetyl CoA. CS activity was measured in a reaction containing 100 mM Tris–HCl, 1 mM MgCl2, 1 mM EDTA, 0.2 mM dithio‐bis (2‐nitrobenzoic acid), 0.3 mM acetyl CoA and 0.5 mM oxaloacetate, pH 8.2. The rate of change in absorbance was monitored at 340 nm (ɛ = 13.6 μmol mL‐1).Activities of the enzymes beta‐hydroxyacyl‐CoA dehydrogenase (OHADH), citrate synthase (CS) and creatine kinase (CK) that participate in fatty acid metabolism and citric acid cycle (CAC) were analysed in the myocardium. LV samples were homogenized 1:20 (wt/vol) in 50 mM Tris–HCl, 1 mM EDTA and protease inhibitor cocktail, pH 7.4, using a Bullet Blender storm, (Next Advanced). The lysate was centrifuged at 12000 2.102, 0.8 mM EDTA, 1.5 mM KCl, 4 mM 2 mercaptoethanol, 0.4 mM NADP+, 1 m mM glucose, 1.4 units glucose‐6‐phosphate dehydrogenase, 0.05% Triton X‐100, pH 7.2. The total assay volume was 165 μL and 10 μL of fresh homogenate was added. The assay was initiated by the addition of glucose and monitored at 340 nm.ATP content was measured as previously.2.11anova) followed by Bonferroni post‐hoc test (parametric distribution) or Kruskal–Wallis followed by Dunn's  was used to analyse differences between groups. Goodman test was used to test differences in HF features. The correlation matrix was generated using a two‐tailed Pearson's correlation test.Data are expressed as mean ± standard deviation or median and percentiles. Kolmogorov–Smirnov normality test was used to test data normal distribution. Two‐way analysis of variance  and sensors of the cellular metabolic stress . In addition, HIF1α was found negatively correlated with the lipid metabolism pathway , which suggests a high relationship between hypoxia and energy perturbation during HF.As expected, angiogenesis was positively correlated with metabolic parameters; VEGF with Heatmap also showed a positive correlation of proteins linked to FA transport and oxidation  with systole . Conversely, metabolism was inversely correlated with cardiac structural and diastole, except by SIRT1 which showed a positive correlation with hypertrophy and diastolic dysfunction indicators. Based on these results, lipid energy metabolism is primarily linked to systolic performance, while FAO decline occurs in parallel with pathological hypertrophy and diastolic dysfunction.4Searching for the therapeutic approaches for the management of heart diseases remains a challenge and non‐pharmacological strategies are emerging as an effective tools for prevention and treatment. Here, we showed that (1) early exercise training had a protective effect against AS‐induced pathological remodelling and cardiac dysfunction in rats, (2) corrected vascular rarefaction in the myocardium and (3) re‐established expression and activity of components of the myocardial lipid metabolism pathway during HF. These benefits were accompanied by a significant alleviation of the HF profile including functional capacity maintenance.For this report, we adapted a training protocol from previous studies that found benefits in functional capacity and cardiac function with different experimental models of cardiac disease.Previous studies attempt to rescue deficient cardiac function in aortic stenosis rats using a late exercise program that resulted in inconsistent conclusions.Regarding cardiac structure, our data demonstrate that AST rats displayed alleviation of the hypertrophic process as visualized in the weight of chambers and echocardiogram parameters. Following the effect on cardiac structure, both diastolic, and systolic impairment were significantly attenuated by ET. Additionally, exercise reduced the occurrence of tachypnoea, pleural effusion, atrial thrombi, liver congestion and ascites, classical symptoms of HF observed in experimental trialsWe mainly aimed to investigate the underlying mechanisms involved with the benefits of exercise on the failing heart. Several studies have documented that transition from hypertrophy towards HF is associated with myocardial capillary rarefaction,Our data reveal that the preservation of VEGF levels and capillary density in hearts of AST occurred in parallel with reduced HIF1‐α protein content compared to the untrained AS group, which presented HIF1‐α overexpression. A recent study conducted in our laboratory found a time‐dependent increase in HIF1‐α levels during the worsening of cardiac function.Based on the above findings related to angiogenesis and hypoxia, we tested whether myocardial lipid metabolism is also associated with cardioprotection promoted by early exercise. This approach is essential since increasing evidence demonstrates that energy metabolic derangements contribute to the pathogenesis of cardiac functional decompensation,To the best of our knowledge, we provide the first scientific literature about the effects of early ET on cardiac lipid metabolism in AS‐induced HF. Pathological hypertrophy was accompanied by a disturbance in the FA utilisation pathway; AS rats presented a reduced expression of key components involved with fatty acid transport and β‐oxidation. Conversely, the impact of PO on the FA pathway was less important in the exercised group, as visualized in the results session, which suggests that ET in course of HF attenuates the pathological pattern of energy generation from FA. In line with present data, the downregulation of genes involved in FA metabolism, from uptake to cleavage within the mitochondrial β‐oxidation cycle, is widely shown.The mechanisms associated with the decreased FAO pathway appear to be related to a transcriptional disorder that includes transcription factors, especially PPARα.Loss of ATP and phosphocreatine are classical characteristics of the failing heart.It was demonstrated a positive correlation between angiogenesis and FA signalling, as well as a negative correlation between cardiac remodelling with both angiogenesis and metabolism, endorsing our results. In summary, our data suggest that early exercise rescues capillary density and attenuates the disturbance of the FAO pathway together with amelioration of cardiac performance, even in the setting of severe hypertrophy. An overview of the results are presented in the Figure 5In our study, we provide an initial analysis of the lipid metabolism since the static measurement of enzymes does not specifically indicate a change in fatty acid oxidation. Additional studies will be needed to deepen the knowledge of the substrate flux and tissue oxygen availability of the hypertrophied heart under early exercise. Also, we did not study the interaction between SIRT1 and RXR, therefore, our data are limited to supposing a plausible mechanism. Regarding energy production, we did not measure the phosphocreatine content, which is found to be initially reduced in heart failure and could reveal energy depletion.6In conclusion, early exercise attenuated cardiac remodelling, reduced HF symptoms and improved the functional capacity of aortic stenosis rats. For these results, we demonstrate that early exercise triggers a consistent effect on the management of AS‐induced HF. Regarding the underlying mechanisms involved with the beneficial effects of ET in AS rats, we raise the assumption that attenuation of cardiac dysfunction potentially is linked to the preservation of lipid energy metabolism, which in turn is related to angiogenesis restoration.Sérgio Luiz Borges de Souza: Conceptualization ; data curation ; formal analysis ; funding acquisition ; investigation ; methodology ; project administration ; resources ; software ; supervision ; validation ; visualization ; writing – original draft ; writing – review and editing . Gustavo Augusto Ferreira Mota: Conceptualization ; investigation ; methodology ; resources ; supervision ; visualization . Vitor Loureiro da Silva: Conceptualization ; investigation ; methodology . Danielle Fernandes Vileigas: Methodology . Paula Grippa Sant'Ana: Methodology . Cristina Schmitt Gregolin: Methodology . Rebeca Lopes Figueira: Methodology . Sabrina Setembre Batah: Methodology . Gilson Masahiro Murata: Conceptualization ; investigation ; resources ; supervision . Silmeia Bazan: Methodology . Alexandre Fabro: Conceptualization ; resources ; supervision . Marina Politi Okoshi: Resources ; visualization . Antonio Carlos Cicogna: Conceptualization ; data curation ; formal analysis ; funding acquisition ; project administration ; supervision ; validation ; visualization ; writing – original draft ; writing – review and editing .The authors declare that there are no conflicts of interest regarding the publication of this article.All authors gave consent for manuscript publication.Appendix S1.Click here for additional data file.


To investigate whether brain volumes were reduced in children aged 6 to 8 years without cerebral palsy, who underwent therapeutic hypothermia for neonatal hypoxic‐ischaemic encephalopathy (patients), and matched controls, and to examine the relation between subcortical volumes and functional outcome.We measured regional brain volumes in 31 patients and 32 controls (median age 7 years and 7 years 2 months respectively) from T1‐weighted magnetic resonance imaging (MRI). We assessed cognition using the Wechsler Intelligence Scales for Children, Fourth Edition and motor ability using the Movement Assessment Battery for Children, Second Edition (MABC‐2).p < 0.05). Differences in subcortical grey‐matter volumes were not independent of total brain volume (TBV). In patients, hippocampal and thalamic volumes correlated with full‐scale IQ  and MABC‐2 total score  independent of age, sex, and TBV. No significant correlations were found in controls. In patients, cortical injury on neonatal MRI was associated with reduced volumes of hippocampi (p = 0.001), thalami (p = 0.002), grey matter (p = 0.015), and white matter (p = 0.013).Patients had lower volume of whole‐brain grey matter, white matter, pallidi, hippocampi, and thalami than controls (false discovery rate‐corrected Children who underwent therapeutic hypothermia have reduced whole‐brain grey and white‐matter volumes, with associations between hippocampal and thalamic volumes and functional outcomes. BGTbasal ganglia and thalamiHIEhypoxic‐ischaemic encephalopathyMABC‐2Movement Assessment Battery for Children, Second EditionPLICposterior limb of internal capsuleTBVtotal brain volumePatients who underwent therapeutic hypothermia for neonatal hypoxic‐ischaemic encephalopathy had lower whole‐brain white‐ and grey‐matter volumes than controls.These patients had smaller pallidi, hippocampi, and thalami.Subcortical volume differences were not independent of total brain volume.Cognitive and motor scores correlated with hippocampal and thalamic volumes in the patients.What this paper addsTherapeutic hypothermia is now standard care for neonatal hypoxic‐ischaemic encephalopathy (HIE) secondary to perinatal asphyxia in high‐income countries and has been shown to reduce the risk of death and severe disability compared with normothermia management.Despite the benefits of therapeutic hypothermia, we have previously demonstrated that early school‐age children who underwent therapeutic hypothermia for HIE and did not develop CP have altered brain structural connectivityIt is well established that the acute hypoxic‐ischaemic insult preceding HIE leads to characteristic patterns of brain injury on neonatal magnetic resonance imaging (MRI) scans, particularly to the deep grey‐matter structures including the hippocampus, thalamus, and basal ganglia.It is not known whether children who have been treated with therapeutic hypothermia for HIE and who do not develop CP have lower volumes of subcortical structures and whether these subcortical volumes are associated with motor and cognitive outcomes. Therefore, we compared the volumes of brain tissues and subcortical structures between early school‐age children cooled for HIE, without CP, and control children matched for age, sex, and socioeconomic status. Second, we examined the relation between subcortical volumes and motor and cognitive outcomes. Finally, we investigated the association between qualitative scores of regional brain injury on neonatal MRI and regional brain volumes at early school‐age.This study was conducted at the Clinical Research and Imaging Centre (CRiCBristol), University of Bristol, UK, with approval from the North Bristol Research Ethics Committee (REC ID 15/SW/0148). Participants' assent was ensured at all times and informed and written consent was obtained from the parents of participants. Patients were sequentially selected from the cohort of children who received therapeutic hypothermia between October 2007 and November 2012 under a standard protocol for perinatal asphyxia‐induced moderate to severe encephalopathy confirmed by amplitude‐integrated electroencephalogram (EEG) assessment.Patients were aged 6 to 8 years and did not have a diagnosis of CP at 2 years based on assessment of motor function and neurological examination . Absence of CP at 6 to 8 years was confirmed using a standard clinical neurological examination including assessment of tone, motor function, and deep tendon reflexes. We excluded children who (1) were cooled outside the standard criteria; (2) were born before 35 weeks' gestation; (3) had an additional diagnosis, for example a metabolic disorder; or (4) did not have English as their primary spoken language.Patients underwent neonatal MRI, which was qualitatively assessed, by an experienced perinatal neurologist (FC) for the presence and extent of brain injury. This was quantified, in the basal ganglia and thalami (BGT), white matter, and cortex (each on a score of 0–3), and the posterior limb of internal capsule (PLIC) (score 0–2), where a higher number indicates more severe injury.We recruited controls from schools around Bristol. Schools were excluded where a patient was currently attending, to protect participants' confidentiality. We included children who were born after 35 weeks' gestation, had not had perinatal asphyxia with HIE, and spoke English as their primary spoken language. To minimize the chance of recruiting children whose parents had concerns (e.g. about their development), parents were made aware that results of children's individual assessments would not be made available to them.www.gov.uk/government/statistics/english‐indices‐of‐deprivation‐2019). The 1 to 10 scale (where 10% of neighbourhoods are assigned to each number) is determined for a given neighbourhood from seven domains of deprivation including income, employment, education, health, crime, barriers to housing and services, and living environment, indicating the decile within which the local area is ranked in the country, from most deprived (1) to least deprived (10). We assessed the index of multiple deprivation for each participant on the basis of postcode at birth.Socioeconomic status was assessed using the index of multiple deprivation as defined for England by the UK Government (Psychologists (led by JT), blinded to patient–control status, assessed the cognitive abilities of the children using the Wechsler Intelligence Scale for Children, Fourth EditionAll children were scanned using a 3 tesla Siemens Magnetom Skyra  and a receive‐only 32‐channel head coil. Following acquisition of localizer images, a T1‐weighted volumetric scan was obtained with the magnetization‐prepared rapid acquisition gradient echo sequence using the following parameters: echo time 2.19 ms; inversion time 800 ms; repetition time 1500 ms; flip angle 9°; field of view 234 mm × 250 mm; 176 slices; 1.0 mm isotropic voxels; generalized autocalibrating partially parallel acquisitions acceleration factor 4.http://fil.ion.ucl.ac.uk/spm)http://www.neuro.uni‐jena.de/cat)https://fsl.fmrib.ox.ac.uk/fsl), to obtain the whole‐brain volume of each tissue type. Whole‐brain grey‐matter volume included both cortical and subcortical grey matter. Total brain volume (TBV) was then calculated as the sum of grey‐ and white‐matter volumes.A brain tissue mask was created for each participant's T1‐weighted data using either SPM8‐VBM , hippocampus, and thalamus, were segmented from each individual's T1‐weighted scan using FSL's automated subcortical segmentation tool, FIRST,t‐test as these variables were normally distributed. One‐way analysis of covariance (ANCOVA) was then used to compare subcortical volumes between groups while controlling for TBV. To examine the association between regional brain volumes and functional outcome, we then assessed the partial Pearson correlation between subcortical volumes, full‐scale IQ score, and MABC‐2 total score, with age, sex, and TBV included as covariates, in the patient and control groups separately. To test whether there was any association, in patients, between qualitative injury scores on neonatal MRI and regional volumes at 6 to 8 years, we performed Wilcoxon rank‐sum tests between the regional volumes of patients with no injury (score = 0) and those of patients with any injury (score >0) for each injury category . In all tests, correction for false discovery rate was applied and p < 0.05 was considered significant. Statistical analysis was performed using IBM SPSS version 24 .Normality was tested with a Q–Q plot and Shapiro–Wilk test. Grey matter, white matter, cerebrospinal fluid, and subcortical grey‐matter volumes were compared between the patient and control groups using a two‐tailed p = 0.040), indicating that the included cohort on average was in poorer condition at 10 minutes of age.Study recruitment is summarized in Figure p = 0.003) and white matter (p = 0.026). Additionally, patients compared with controls had significantly lower volumes of pallidi (p = 0.026), hippocampi (p = 0.004), and thalami (p = 0.013). When taking differences in TBV into account, there were no significant differences in the volumes of subcortical structures .Figure r = 0.477, p = 0.010) and thalami . Additionally, MABC‐2 total score significantly correlated with the volume of the hippocampi  and thalami . In controls, MABC‐2 total score correlated with the volume of the putamen  and hippocampi , but neither of these was significant after correction for false discovery rate .Correlations between subcortical volumes and functional outcome are shown in Figure q > 0.05); however, the sample sizes for these injury sites were very small. Patients with cortical injury scores greater than 0 on neonatal MRI had significantly reduced hippocampi (p = 0.001), thalami (p = 0.002), grey matter (p = 0.015), and white matter (p = 0.013) compared with patients with no cortical injury. When TBV was included as a covariate in an ANCOVA, hippocampal volume was still associated with cortical injury (p = 0.002), but the association with thalamic volume was not significant after correction for false discovery rate. Regional brain volumes at school‐age are plotted against scores from qualitative scores of injury on neonatal MRI in Figures Median qualitative scores of injury on neonatal MRI in patients were low, as expected for this group, which did not include those with severe disability or any CP. There was no association between regional volumes at school‐age and qualitative injury scores on neonatal MRI for the white matter, BGT, or PLIC  are associated with later adverse neurodevelopmental outcomes.We found no association between regional brain volumes at school‐age and injury patterns in the BGT, PLIC, or white matter on neonatal MRI. This may suggest cortical injury is more sensitive than other sites of injury in contributing to altered structural brain development in cooled children without severe disability; however, owing to the small sample sizes in these tests, particularly for the PLIC, BGT, and white‐matter associations, we cannot draw firm conclusions from these analyses.Owing to the difficulties associated with scanning children of this age group, the MRI data from many participants were not of sufficient quality to be included in the analysis. A larger sample could have enabled more sensitive detection of group differences and correlations, particularly as this cohort represented those with non‐severe outcomes and may therefore be characterized by very subtle differences in brain structure. Specifically, a larger cohort would allow more robust analysis of differences in regional brain volumes between patients with and without injury on neonatal MRI. Additionally, to increase statistical power and reduce the effect of multiple comparisons, we did not investigate associations with subscales of the cognitive and motor assessments. This is largely because few studies have investigated subcortical grey‐matter volumes in cooled children with non‐severe outcomes, so we opted for a more exploratory, rather than hypothesis‐driven, approach. Future studies could focus on the involvement of the hippocampus and thalamus in domains of cognitive and motor performance.We have demonstrated that early school‐age children cooled for HIE, without CP, had altered regional brain volumes compared with matched controls. Further investigation is required to determine neuroprotective strategies or therapeutic interventions that improve outcomes.The Baily Thomas Charitable Fund (TRUST/VC/AC/SG4681‐7596), David Telling Charitable Trust, Sparks (05/BTL/01 and 14/BTL/01), and Moulton Foundation. A.P.C.S. is supported by the Wellcome Trust (WT220070/Z/20/Z). J.C.W.B. is supported by the UK Medical Research Council (MR/N026969/1).Table S1: Criteria for qualitative assessment of the presence and extent of brain injury on neonatal MRI.Click here for additional data file.Table S2: Regional volume for patients and controls.Click here for additional data file.Table S3: Regional volume at school‐age for patients grouped by white matter injury scores on neonatal MRI.Click here for additional data file.Table S4: Regional volume at school‐age for patients grouped by basal ganglia and thalamus injury scores on neonatal MRI.Click here for additional data file.Table S5: Regional volume at school‐age for patients grouped by posterior limb of internal capsule injury scores on neonatal MRI.Click here for additional data file.Table S6: Regional volume at school‐age for patients grouped by cortex injury scores on neonatal MRI.Click here for additional data file.Figure S1: Study recruitment.Click here for additional data file.Figure S2: Plots showing the distribution of volumes, measured from MRI at 6–8 years, with patients grouped by scores from neonatal MRI assessment of white matter.Click here for additional data file.Figure S3: Plots showing the distribution of volumes, measured from MRI at 6–8 years, with patients grouped by scores from neonatal MRI assessment of basal ganglia and thalami.Click here for additional data file.Figure S4: Plots showing the distribution of volumes, measured from MRI at 6–8 years, with patients grouped by scores from neonatal MRI assessment of posterior limbs of the internal capsule.Click here for additional data file.Figure S5: Plots showing the distribution of volumes, measured from MRI at 6–8 years, with patients grouped by scores from neonatal MRI assessment of the cortex.Click here for additional data file.


Previous pilot studies suggest that sodium benzoate may be a potential cognitive enhancer for patients with Alzheimer's disease (AD), schizophrenia, or late‐life depression. Especially for AD treatment, a confirmatory trial with predictive biomarkers is urgently needed. This study aimed to confirm benzoate as a novel treatment for AD and to discover its optimal dose and biomarkers.A 24‐week, dose‐finding, randomized, double‐blind, placebo‐controlled trial, with clinical measurements at weeks 0, 8, 16, and 24, was conducted in three major medical centers in Taiwan. Among 154 patients screened for AD, 149 were eligible and randomized to one of the four treatments: (i) benzoate 500 group (fixed 500 mg/day); (ii) benzoate 750 ; (iii) benzoate 1000 ; and (iv) placebo. The primary outcome measure was AD assessment scale‐cognitive subscale (ADAS‐cog).P = 0.026 at week 24), with female advantage. Higher plasma catalase at baseline predicted better outcome. Benzoate receivers tended to have higher catalase and glutathione than placebo recipients after treatment. The four intervention groups showed similar safety profiles.The benzoate 1000 group performed best in improving ADAS‐cog (By enhancing two vital endogenous antioxidants, catalase and glutathione, sodium benzoate therapy improved cognition of patients with AD, with higher baseline catalase predicting better response. Supporting the oxidative stress theory, the results show promise for benzoate as a novel treatment for AD. Dementia is gaining increasing attention because of the high mortality and morbidity, which needs collaborative care.In addition to AChEIs, NMDAR antagonists have been developed to treat AD based on the ‘glutamate excitotoxicity theory’ for the middle‐late phase of AD.in vitro and in vivo evidence.n = 60),Sodium benzoate may play a neuroprotective role through reducing oxidative stress, based upon n = 149) and to explore the prospective biomarkers. Glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD) are three first‐line endogenous antioxidants, which prevent and reduce oxidative stress.The current study aimed to find out the optimal dose of benzoate for the treatment of AD in a larger sample of patients , conducted by three major medical centers, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan, China Medical University Hospital, Taichung, Taiwan, and Tsaotun Psychiatric Center, Nantou, Taiwan, and approved by the Institutional Review Board of Chang Gung Memorial Hospital, Taiwan (201405724A3C504), Institutional Review Board of China Medical University Hospital, Taiwan (CMUH104‐REC2‐090) and Institutional Review Board of Tsaotun Psychiatric Center, Taiwan (104039), and conducted in accordance with the current revision of the Declaration of Helsinki.This 24‐week, randomized, double‐blind, placebo‐controlled trial was registered on the ClinicalTrials.gov  were aged 50–100; (ii) satisfied National Institute of Neurological and Communicative Disorders and Stroke and the AS and Related Disorders Association (NINCDS‐ADRDA)Exclusion criteria included: (i) Hachinski Ischemic Score > 4; (ii) substance abuse/dependence; (3) Parkinson disease, epilepsy, dementia with psychotic features; (iv) major depressive disorder; (v) major physical illnesses; and (vi) severe visual or hearing impairment.All eligible patients were randomly allocated to four groups: (i) benzoate 500 group (fixed dose at 500 mg/day); (ii) benzoate 750 group ; (iii) benzoate 1000 group ; and (iv) placebo group. Two hundred and fifty milligrams of sodium benzoate or placebo were packed with identical capsules provided in coded containers. Patients were randomized through a computer‐generated randomization table to receive placebo or benzoate treatment in a 1:1:1:1 ratio. Non‐blinded pharmacists dispensed appropriate medication for treatment according to the randomization table. Patients, caregivers, and investigators, except the investigational pharmacist, were all blind to the assignment. Patient medical adherence and safety were closely monitored by caregivers and research physicians, and pill‐counting was monitored by the study staff.During the study, limited use of benzodiazepines (up to 4‐mg/day lorazepam) was allowed as concomitant medication for agitation or insomnia. In case of side‐effect intolerance or clinical worsening, the patients could be withdrawn earlier.The primary outcome measure was the Alzheimer's Disease Assessment Scale‐cognitive subscale (ADAS‐cog)The secondary outcome measurements included Clinician's Interview‐Based Impression of Change plus Caregiver Input (CIBIC‐plus)The CIBIC‐plus is a global assessment of change based on a comprehensive, semi‐structured interview that includes caregiver‐supplied information. It is a seven‐point rating scale ranging from 1 to 7, where one represents markedly improved; four represents no change; and seven represents markedly worse.T scores of the three cognitive domains . The raw score of each cognitive domain was standardized to a T score with a mean of 50 and a SD of 10 for making each test comparative. We have successfully utilized it in the previous study on early‐phase AD.The battery of additional cognitive tests included speed of processing (category fluency), working memory ,Systemic adverse effects were evaluated every 8 weeks  by physical and neurological examinations and laboratory tests including complete blood count (CBC) and biochemistry and reviewed by applying the Udvalg for Kliniske Undersogelser Side‐effects Rating Scale.Only raters who reached the intra‐class correlation coefficients of ≥0.90 during pre‐study training were allowed to rate the study patients. To maintain high inter‐rater reliability and to prevent rater drift, raters met at least once per quarter for training and reliability retesting. To minimize inter‐rater variability, each individual patient was assessed by the same raters throughout the trial.DAAO and three antioxidants in peripheral blood were measured using the commercially available kits at baseline and endpoint.DAAO protein levels in serum were assayed with the kit according to the manufacturer's recommended protocol . The detailed method has been described elsewhere.GSH levels in plasma were measured using the kit according to the manufacturer's recommended protocol . The detailed method has been described elsewhere.SOD levels in plasma were analyzed using the kit according to the manufacturer's recommended protocol . The detailed method has been described elsewhere.CAT in plasma was measured using the kit according to the manufacturer's recommended protocol . The detailed method has been described elsewhere.All of the aforementioned analyses were repeated twice.n = 30 in each group),Based on our previous trial in early‐phase AD  was applied to compare differences of continuous variables between groups. To compare the changes from baseline in repeated‐measure assessments, we used the generalized estimating equation (GEE) method's multiple linear regression models with treatment, visit, and treatment‐visit interaction terms after adjusting the baseline value of the outcome measure. The working correlation matrix was specified as autoregressive of order 1.Chi‐square test (or Fisher's exact test) was used to compare differences of categorical variables and one‐way analysis of variance  for continuous variables among four treatment groups. The Subgroup analysis was also performed based on the previously reported gender difference  in benzoate treatment for the behavioral and psychological symptoms of dementia (BPSD).Multiple linear regression analyses were used to generate predictive models for treatment response.P‐values for clinical measures were based on two‐tailed tests with a significance level of 0.05.All data were analyzed by SPSS version 22.0 . All n = 2), inadequate severity at baseline (n = 1), and inability to follow protocol (n = 2). One hundred and forty nine patients were eligible and randomly allocated into four treatment groups  at baseline were similar among the four groups  , unsatisfactory therapeutic response , and physical problems   patients in the benzoate 500 group, 29 (80.6%) in the benzoate 750 group, 30 81.1%) in the benzoate 1000 group, and 33 (86.8%) in the placebo group completed the 24‐week trial , while the benzoate 750 group also showed improvement in ADAS‐cog   and endpoint (P = 0.11) (not shown). Of note, the sample size in men  was inadequate. Therefore, the subgroup analyses focused on the female group .As shown in Figure Z = −2.24, P = 0.025), week 16 , week 24 , and endpoint ; the benzoate 1000 group displayed a trend (insignificant) improvement in ADAS‐cog at week 24 (P = 0.086) and endpoint (P = 0.082); and the benzoate 500 group did not differ significantly from the placebo group in improving ADAS‐cog at each visit (not shown).Regarding the dose difference among the female patients, the benzoate 750 group, compared to the placebo group, improved significantly in ADAS‐cog at week 8 . The benzoate 1000 group showed a trend (insignificant) superiority over the placebo group in CIBIC‐plus at endpoint . The benzoate 500 and the placebo groups were similar in CIBIC‐plus performance (not shown).The subgroup analysis for the secondary outcome measures focused on the female group, because, as aforementioned, the sample size of the male group appeared too small. Among the female patients, the benzoate 750 group excelled the placebo group in CIBIC‐plus at endpoint .The four treatment groups showed similar performances in adverse effects, as measured by Udvalg for Kliniske Undersøgelser (UKU) Side‐effects Rating ScaleAll laboratory parameters were measured at baseline and endpoint Table . At baseP = 0.07, Table P = 0.29, Table After treatment, the difference in plasma CAT among the four treatment groups did not reach significance  among the benzoate recipients Table .P = 0.001, Table P = 0.012, Table Baseline plasma CAT was correlated with ADAS‐cog improvement after treatment  in the benzoate‐treated group, not in the placebo group.Moreover, differences in the level of CAT were correlated with the magnitudes of ADAS‐cog improvement (score reduction from baseline to endpoint) (n = 60),n = 149) further confirmed benzoate as a novel therapeutic approach for AD.The main findings of this benzoate dose‐finding, randomized, double‐blind, placebo‐controlled trial for AD included: (i) 1000 mg of benzoate led to the best outcome, while 750 mg/day also generated similar efficacy to 1000 mg/day Table ; (ii) woIn addition to a potential DAAO inhibitor,in vitro,Blood DAAO levels were found to be related with cognitive aging in both cross‐section and prospective studies.via augmenting two vital endogenous antioxidants, CAT and GSH. Earlier, benzoate was also able to increase plasma CAT in treatment‐resistant schizophrenia patients; importantly, the CAT increment was positively related with clinical improvement.Supporting the oxidative stress theory of AD,Sodium benzoate's oxidative‐stress reduction actitivityFor detailed comparisons, in the previous pilot AD trial,In terms of the safety domain, the current study found that all three doses  of benzoate and placebo had similar performance Table . In accoP‐value for the primary outcome but necessary to correct the P‐values for the secondary outcomes . If we took all outcomes simultaneously, the significancy of the primary outcome would have become insignificant after correcting the P‐value. Therefore, future clinical trials with larger sample sizes are needed. The second limitation is the 24‐week treatment duration. It remains unclear whether a longer‐term (such as 48 weeks) treatment of benzoate can improve the cognitive and general function. Thirdly, whether the finding in Han Taiwanese can be extrapolated to other populations requires further studies. Fourthly, we did not measure DAAO, D‐serine, and the three antioxidants in the brain, and the blood–brain relationships of these potential biomarkers remain unknown in the participants. Moreover, a recent studyThis study was limited by its moderate sample size, which might have led to underpowered results. Further, regarding multiplicity adjustments, this study had one primary outcome (ADAS‐cog) and two secondary outcomes . If we considered the primary and secondary outcomes separately, it would have been dispensable to correct the via augmenting two vital endogenous antioxidants, CAT and GSH, shows promise as a novel treatment for AD. Women and the patients with higher plasma CAT before treatment were associated with better treatment outcome. If these findings can be reconfirmed in future larger‐sized studies in other populations, this innovative approach for AD will instill hope for the rapidly growing dementia population.This study suggests that sodium benzoate, All authors report no biomedical financial interests or potential conflicts of interest.C.‐H.L. had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Concept and design: C.‐H.L., H.‐Y.L. Acquisition, analysis, or interpretation of data: C.‐H.L., S.‐H.W., H.‐Y.L. Drafting of the manuscript: C.‐H.L., H.‐Y.L. Statistical analysis: C.‐H.L., S.‐H.W. Obtained funding: C.‐H.L., H.‐Y.L. Administrative, technical, or material support: C.‐H.L., H.‐Y.L. Supervision: C.‐H.L., H.‐Y.L.This work was supported by the National Health Research Institutes, Taiwan ; the Ministry of Science and Technology in Taiwan ; the Chang Gung Memorial Hospital, Taiwan , and the China Medical University Hospital, Taiwan (DMR‐HHC‐111‐9). The sponsors were not involved in the design and conduct of the study; collection, management, analysis, and interpretation of the data; and preparation, review, or approval of the manuscript.This study was approved by the Institutional Review Board of Chang Gung Memorial Hospital, Taiwan (201405724A3C504), Institutional Review Board of China Medical University Hospital, Taiwan (CMUH104‐REC2‐090) and Institutional Review Board of Tsaotun Psychiatric Center, Taiwan (104039), and conducted in accordance with the current revision of the Declaration of Helsinki.Table S1. Results of measures of secondary outcomes over the 24‐week treatment using generalized estimating equations (GEE) method, which simultaneously compared the four treatment groups using a single analysis.Table S2. Treatment‐emergent adverse events during the study.Table S3. Measures of DAAO and antioxidants over the 24‐week treatment of the four treatment groups.Click here for additional data file.


Unhealthy alcohol use significantly contributes to viral non‐suppression among persons with HIV (PWH). It is unknown whether brief behavioural interventions to reduce alcohol use can improve viral suppression among PWH with unhealthy alcohol use in sub‐Saharan Africa (SSA).As part of the SEARCH study (NCT04810650), we conducted an individually randomized trial in Kenya and Uganda of a brief, skills‐based alcohol intervention among PWH with self‐reported unhealthy alcohol use  and at risk of viral non‐suppression, defined as either recent HIV viral non‐suppression (≥400 copies/ml), missed visits, out of care or new diagnosis. The intervention included baseline and 3‐month in‐person counselling sessions with interim booster phone calls every 3 weeks. The primary outcome was HIV viral suppression (<400 copies/ml) at 24 weeks, and the secondary outcome was unhealthy alcohol use, defined by AUDIT‐C or phosphatidylethanol (PEth), an alcohol biomarker, ≥50 ng/ml at 24 weeks.Between April and September 2021, 401 persons  were enrolled from HIV clinics in Uganda (58%) and Kenya (27%) and alcohol‐serving venues in Kenya (15%). At baseline, 60% were virally suppressed. Viral suppression did not differ between arms at 24 weeks: suppression was 83% in intervention and 82% in control arms . Among PWH with baseline viral non‐suppression, 24‐week suppression was 73% in intervention and 64% in control arms . Unhealthy alcohol use declined from 98% at baseline to 73% in intervention and 84% in control arms at 24 weeks . Effects on unhealthy alcohol use were stronger among women  than men  and among participants with a baseline PEth⩽200 ng/ml  versus >200 ng/ml .In a randomized trial of 401 PWH with unhealthy alcohol use and risk for viral non‐suppression, a brief alcohol intervention reduced unhealthy alcohol use but did not affect viral suppression at 24 weeks. Brief alcohol interventions have the potential to improve the health of PWH in SSA by reducing alcohol use, a significant driver of HIV‐associated co‐morbidities. Sub‐SahHealth services addressing alcohol consumption are limited in SSA , 13. OnlWe conducted a randomized controlled trial (RCT) to compare the effectiveness of a brief, culturally adapted , 22 skil22.1During the pilot phase (Phase A) of the SEARCH‐SAPPHIRE trial (NCT04810650), we conducted a two‐arm, individual RCT of the impact of a brief, culturally adapted skills‐based alcohol counselling intervention on HIV viral suppression among PWH with heavy alcohol use and at risk for viral non‐suppression.2.2Between April and September 2021, we screened participants at rural government‐run health centres in three communities in southwestern Uganda and five communities in Western Kenya and at alcohol‐serving venues in the Kenyan study communities. In Uganda, COVID‐19 restrictions closed all bars and nightclubs from March 2020 until January 2022, precluded recruitment at these sites.2.3For clinic‐based recruitment, the study team performed chart and registry reviews and met with clinic staff to pre‐identify adults potentially eligible for the study. Venue‐based recruitment used methods developed previously . Briefly2.4The study inclusion criteria were ≥18 years of age, AUDIT‐C (modified to prior 3 months)  positive2.5We randomized participants 1:1 to the alcohol counselling intervention or standard‐of‐care (control) using stratified block randomization (block size = 2 and 4), computer‐generated and stratified by country. Participants were not masked to the randomization arm, but the study statistician (LBB) was masked until trial completion.2.6At baseline, we administered a structured questionnaire which included demographics, AUDIT‐C adapted to the prior 3 months, ART use, and current tobacco and other substance use assessments. We completed baseline study visits within 14 days of enrolment. After 24 weeks from baseline, study staff administered the AUDIT‐C for the prior 3 months.Laboratory assessments included HIV‐1 RNA viral load, CD4 cell count and PEth level, a quantitative biomarker of recent alcohol consumption, at baseline, and viral load and PEth at week 24. We prepared dried blood spots onto Whatman 903 cards, and shipped them in batches to a commercial laboratory, the United States Drug Testing Laboratory , for quantification of PEth (16:0/18:1 homologue), with an 8 ng/ml lower limit of quantification . PEth coParticipants received a transport reimbursement  for in‐person study visits at baseline, week 12 (intervention only) and week 24.2.7The intervention was a multi‐session brief alcohol counselling intervention based on the Information, Motivation, and Behavioral skills (IMB) model  and implThe intervention included two in‐person counselling sessions, performed at baseline and week 12, with brief “booster” phone‐based counselling session performed every 3 weeks between in‐person sessions. The in‐person sessions were workbook‐guided and designed to reflect on the consequences of alcohol use on one's health and HIV treatment, identify risky moods and situations that precede alcohol use, build skills for alternative behaviours and set individualized goals to decrease alcohol intake. Booster calls were tailored to participants’ goals and designed to check progress and provide positive reinforcement and encouragement as appropriate. Intervention participants received viral load and adherence counselling based on results from the baseline study visit by study staff at the 3‐week phone call and by clinic staff at their post‐baseline regular clinic visit.Counselling was conducted by lay counsellors trained by a licensed clinical psychologist (SWK) on the basic Motivational Interviewing skills and implementation of the manualized intervention. SWK provided ongoing group supervision for the counsellors.2.8Participants randomized to the control arm received brief advice on the harmful effects of alcohol and safe levels of drinking at the baseline visit in line with the country standard of care, viral load feedback and adherence counselling at their post‐baseline regular clinic visit, and an invitation to receive the intervention counselling and materials after 48 weeks.Participants in both study arms had access to a toll‐free hotline to reach study staff with questions or problems during the study.2.9n = 5/counsellor)  31–43) years and 286 (71%) had completed primary education or less (Table n = 391) of participants met the definition for unhealthy alcohol use; a subset of participants  reported lower AUDIT‐C scores after enrolment and had baseline PEth <50 ng/ml.The median baseline AUDIT‐C score was 6 (IQR 4–8). The mean baseline PEth was 486.7 ng/ml (95% CI: 423.9–549.6), which is above the recommended cutoff for chronic excessive alcohol use (200 ng/ml) . In 283 3.2Over 24 weeks, 22/400 (6%) participants moved out of study communities (12/197 intervention and 10/203 in control); additionally, one control participant died. Endpoint viral loads were obtained on 371/377 (98%) of the remaining participants: 181/185 (98%) intervention and 190/192 (99%) control. Endpoint PEth was obtained in 181/185 (98%) intervention and 188/192 (98%) control participants.3.3n = 29 sessions observed) were 93% (IQR 82–94%) and 83% (IQR 79–92%), respectively.Among the 197 intervention arm participants, 196 (99%) completed the baseline in‐person counselling session and 184 (93%) completed session 2. Booster calls were completed for 160 (81%), 148 (75%) and 151 (77%) at 3, 6 and 9 weeks, respectively. Median intervention content fidelity and counselling skills scores  in the intervention and 82% (95% CI: 78–87%) in the control arm at 24 weeks  at 24 weeks in the intervention compared to control. Unhealthy alcohol use declined from 98% at baseline in both arms to 73% (95% CI: 68–78%) in the intervention and 84% (95% CI: 80–89%) in the control, yielding a relative reduction of 14%  are robust and may yield viral suppression even with suboptimal adherence  due to aThird, factors outside of the alcohol counselling intervention may have driven the high viral suppression in both arms. Our study procedures, which identified unhealthy alcohol use through screening, provided brief alcohol counselling in the control, gave viral load feedback and gave transportation reimbursements, coupled with universal ART eligibility may have been sufficient to increase engagement in care and adherence, yielding the increase in viral suppression from 60% to >82% in both arms. Additionally, adaptations to HIV care provision due to COVID‐19 pandemic restrictions and increased provision of differentiated care models , includiOur trial resulted in a significant reduction in unhealthy alcohol use among intervention participants compared to control. This is one of the first alcohol intervention trials, to our knowledge, to incorporate PEth into the alcohol outcome measure. The composite measure of unhealthy alcohol, comprised of self‐report and an alcohol biomarker , helps to overcome limitations in the sensitivity of either measure alone and to reduce bias introduced by potential discrepancies in self‐report and PEth that have been demonstrated in PWH in observational studies in Uganda , 43, 44 Our trial has several limitations. In this individual RCT, there was potential for contamination between trial arms if participants discussed or shared counselling resources , which m5In summary, a brief, skill‐based alcohol counselling intervention offered over 3 months had no effect compared to the control condition on 24‐week viral suppression among PWH engaged in unhealthy alcohol use, despite reducing levels of unhealthy alcohol use. Our findings highlight that PWH with unhealthy alcohol use in SSA can achieve high levels of viral suppression and, with a brief alcohol intervention, can reduce alcohol use, potentially decreasing associated co‐morbidities and improving health overall.The authors report no competing interests.SBP, GC, MLP, DVH, MK and JAH contributed to the study design, data analysis and interpretation, literature search and writing of the manuscript. LBB contributed to the study design, data analysis and interpretation, and writing of the manuscript. FM, FO, JK, JA, AO, EK, GA, EB, SK, DO, MB, JS and SW‐K contributed to the study design, data interpretation and writing of the manuscript. The authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.The study was supported by the U.S. National Institutes of Health NIAID/NHLBI/NIMH U01 AI150510 , NIAAA K23 AA029045 (SBP) and NIAAA K24 AA022586 (JAH).Supporting Information file 2: SEARCH‐Alcohol_SAP_v1.0. Format: Word document. Pre‐specified statistical analysis plan.Text S1: Pre‐specified SAPPHIRE Alcohol Statistical Analysis PlanClick here for additional data file.Supporting Information file 1: CONSORT 2010 Checklist_JIAS. Format: Word document. Standardized CONSORT checklist for randomized controlled trials.Text S2: CONSORT 2010 checklist of information to include when reporting a randomized trial.Click here for additional data file.


Persimmon water kefir had higher total phenolic contents, twice as much as mandarin water kefir (p < .05). Both water kefirs had good color properties. The organoleptic acceptability of the fruit water kefirs was promising.Water kefir is a non‐dairy probiotic beverage. It is obtained by fermentation of water kefir grains with a sugar solution. This study aims to determine the properties of water kefir beverages obtained by fermentation of mandarin and persimmon‐containing water for 42 h. According to microbiological results, both fruits containing water samples provided a high number of lactic acid bacteria and yeasts. Moreover, after fermentation, pH, Brix, and dry matter content did not significantly differ. On the other hand, fructose, maltose, and acetic acid contents of mandarin water kefir are significantly higher than persimmon water kefir ( The organoleptic acceptability of the fruit water kefirs was promising. On the other hand, Citrus fruits can be used to produce functional foods and maintain viability for probiotic microorganisms  is rich in nutrients and bioactive components such as proteins, sugar, vitamin A, vitamin B6, vitamin B12, vitamin D, vitamin C, vitamin E, calcium, potassium, polyphenols, flavonoids, and carotenoids  and dried Persimmon (Diospyros kaki) were purchased from a local market  on March 2021. Water kefir grains were obtained from Danem Dairy Products .Mandarin  sterile water. Water kefir grains were added to prepared mandarin and persimmon samples at a rate of 2% (w/w). Fermentation was carried out at 25°C for 42 h. During the fermentation, pH was measured and recorded Figure . After c2.2.1Lactobacillus spp., Lactococcus spp., and yeasts, respectively. A standard incubation temperature appropriate for each target microorganism was used. Microbiological analysis results were expressed as log CFU mL−1 , M17 Agar (Merck), and PDA (Merck) to enumerate 2.3Total soluble solid contents (Brix) were carried out at 20°C using an Abbe refractometer . pH was obtained by directly measuring fruit‐based water kefir samples using a pH meter  equipped with a glass electrode. pH measurement was done in duplicate, and the experiment was conducted in triplicate during fermentation.2.42 column  at 30°C at a rate of 1.0 mL/min. The injection volume was 20 μL. The solvent employed was a 75:25 mixture of acetonitrile and HPLC‐grade water. The identified sugars were quantified using peak areas and a calibration curve with corresponding standards.Sugar contents  were determined according to the HPLC method of Petkova and Denev  with som2.53PO4/H2O (pH 2.2) at a 0.8 mL/min flow rate was used. The column used was a TR‐016059 Tracer Extrasil ODS2 supplied by Tecknokroma S. Coop. C. Ltd. . Organic acid quantification was based on calibration curves created for each of the compounds identified in the samples. Standard organic acids included acetic acid, citric acid, formic acid, fumaric acid, lactic acid, malic acid, oxalic acid, and tartaric acid.The organic acid measurement was performed according to Guzel‐Seydim et al.  with sli2.6y = 5.5629x + 0.249: R2 = 0.9753).The Folin–Ciocalteu colorimetric method, described by Singleton et al. , was use2.7+ radical cation is generated by reacting 7 mM ABTS+ and 2.45 mM potassium persulfate via incubation at room temperature in the dark for 12–16 h. The ABTS+ solution was diluted with 80% HPLC‐grade ethanol to an absorbance of 0.700 ± 0.040 at 734 nm and equilibrated at 30°C. After introduction of a 30 μL aliquot of each dilution into the assay, it produced from 20 to 80% inhibition of the blank absorbance. To 3 mL of diluted ABTS+, 30 μL of each sample was added and mixed thoroughly. The reactive mixture was allowed to stand at room temperature for 6 min, and the absorbance was recorded immediately at 734 nm. Trolox standard solutions (concentrations from 0 to 2.5 mM) in 80% ethanol were prepared and assayed using the same conditions. Appropriate solvent blanks were run in each assay. The percent of inhibition of absorbance at 734 nm was calculated and plotted as a function of concentration of Trolox for the standard reference data. The absorbance of the resulting oxidized solution was compared to that of the calibrated Trolox standard. The absorbance was recorded at 734 nm. TEAC results have been expressed as mM Trolox equivalents (y = 83.877x + 30.485: R2 = 0.9989).This method was conducted according to Re et al. . Briefly2.8Radical scavenging activity of the water kefir was determined as described by Singh et al. . Differe2.9Y = 92.7, x = 0.3160, y = 0.3321)  was used to determine the color of both fruit water kefir samples. CIE The color space coordinates L* (lightness), a* (red‐green), and b* (yellow‐blue) were recorded using a white standard calibration plate . A group of eight trained panelists (five men and three women ranging in age from 25 to 52 years) conducted the tests to identify five descriptors based on the samples' appearance, taste, aroma, texture, and perception. A sensory panel was assembled, and the organoleptic assessment was graded using a five‐point scale, with five being highly desirable and one being highly undesirable. Fruit water kefir samples were served in opaque plastic cups at 7–10°C and coded with three‐digit numbers.2.11Descriptive statistics  were used to determine the sample characteristics of three replicates. The paired t‐test was used to assess differences in beverage components. SPSS v24.0 was used (IBM). The significance level was set at 0.05.33.1Lactobacilli spp. and Lactococci spp. The lactococci and lactobacilli counts of mandarin and persimmon kefir reached 7.87–7.84 log CFU mL−1 and 8.08–8.16 log CFU mL−1, respectively (Table Elaeagnus angustifolia), apple, quince, grape, kiwi fruit, prickly pear, pumpkin, red pitaya, and pomegranate could be efficient carriers of lactic acid bacteria . During the fermentation, sugars were converted into ethanol and CO2, and thereby the sugar content of water kefir beverages made with fruit juices decreased. Similar results were reported in previous studies for water kefir‐like beverages , while glucose and sucrose contents in MDK and PNK were slightly different (p > .05). Sucrose is one of the main carbohydrates during the fermentation processes of water kefir, and it can be metabolized by three different pathways in many LAB species. The sugar profiles found in this study are comparable to those findings by Bueno et al. , whereas the malic acid was higher in persimmon water kefir . On the other hand, the MDK and PNK had TEAC values of 15.89 and 9.44 mmol TE L−1, respectively (p < .05); however, DPPH values of both fruit‐based kefirs were similar (p > .05). After water kefir fermentation of soy and quinoa protein concentrates, the total phenolic content and antioxidant activity increased significantly . That subjective evaluation of fruit water kefir samples was compared to the results shown in Table Because of its relationship with other physicochemical and sensory properties, color is important for beverages. L*, a*, and b* values of MDK and PNK were reported in Table The yellowness of the persimmon water kefir samples was higher Table . The neg3.72, yeasty aroma, liquorish flavor, and refreshing taste  in taste and acceptability attributes for water kefirs based on fruit types. The acceptability of water kefirs was nearly average for participants. The uniformity in color and flavor was attributed to fermentation. Fruity, acidic, and yeasty taste was identified among sub‐sensory descriptors of water kefirs in this study. Based on this context, grape juice with the addition of water kefir obtained a better acceptance by the panelists concerning color, acidity, aroma, and overall impression, obtained mean scores for all evaluated attributes ranging from 6.76 to 8.35 (Santos et al., In this study, the descriptive words for mandarin and persimmon fruit‐based kefir samples were homogenous texture with bright color, slightly acidic, slightly alcoholic, and presence of COe Figure . There wIt has been stated that the sensory properties of water kefir vary depending on the type and quantity of water kefir culture, fermentation, and storage temperature, the type of fruit or vegetable used in production, and the content of water‐soluble substances (Corona et al., 4Studies on the technological and functional properties of water kefir are important because various fruits greatly impact and differ in product characteristics. Also, fermentation of water kefir grains with alternative substrates is needed to extend the beneficial health effects of water kefir for non‐dairy consumers, vegans, and allergic/lactose intolerant individuals. This study obtained water kefir beverages with high antioxidant activity properties using mandarin and persimmon fruits. Moreover, the microbiological contents of fruit‐based water kefir were very satisfying due to the nourishing environment provided by fruits. Even though the sensory scores of the fruit water kefirs were promising, further studies are necessary to optimize their organoleptic properties.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Glioblastoma (GBM), the most common primary malignant brain tumor, has a poor prognosis, with a median survival of only 14.6 months. The Warburg effect is an abnormal energy metabolism, which is the main cause of the acidic tumor microenvironment. This study explored the role of the Warburg effect in the prognosis and immune microenvironment of GBM.A prognostic risk score model of Warburg effect‐related genes (Warburg effect signature) was constructed using GBM cohort data from The Cancer Genome Atlas. Cox analysis was performed to identify independent prognostic factors. Next, the nomogram was built to predict the prognosis for GBM patients. Finally, the drug sensitivity analysis was utilized to find the drugs that specifically target Warburg effect‐related genes.Age, radiotherapy, chemotherapy, and WRGs score were confirmed as independent prognostic factors for GBM by Cox analyses. The C‐index  and area under curve (>0.7) indicated that the nomogram exhibited excellent performance. The calibration curve also indicates excellent consistency of the nomogram between predictions and actual observations. In addition, immune microenvironment analysis revealed that patients with high WRGs scores had high immunosuppressive scores, a high abundance of immunosuppressive cells, and a low response to immunotherapy. The Cell Counting Kit‐8 assays showed that the drugs targeting Warburg effect‐related genes could inhibit the GBM cells growth in vitro.Our research showed that the Warburg effect is connected with the prognosis and immune microenvironment of GBM. Therefore, targeting Warburg effect‐related genes may provide novel therapeutic options. In this study, a reliable Warburg effect‐related genes risk scoring model was identified. This model has a significantly excellent predictive value for the prognosis of glioblastoma (GBM) patients, suggests their immunotherapy response, and identifies new GBM therapeutic targets. Moreover, we also explored the drugs that target Warburg effect‐related genes and validated them utilizing Cell Counting Kit‐8 (CCK‐8) assays. This study gives a novel tool for the clinical prediction of prognosis in GBM and contributes to the development of new therapeutic strategies.22.1TERT) promoter mutation, epidermal growth factor receptor (EGFR) gene amplification, or +7/−10 copy number changes in isocitrate dehydrogenase (IDH)‐wildtype diffuse astrocytomas and IDH‐wildtype GBM were selected as the training set based on the WHO CNS5. Duplicate patient records were deleted. Lastly, data from defined GBM patients based on WHO CNS5 were included for further analysis. Single‐nucleotide polymorphism (SNP) and copy number variation (CNV) data for each GBM patient were retrieved from the University of California Santa Cruz Xena website. The validation set for this study was obtained after excluding LGG and IDH mutant GBM from the Chinese Glioma Genome Atlas (CGGA) database. The data on gene expression from the training and validation cohorts were log2 transformed [log2 (FPKM+1)]. Fragments per kilobase million (FPKM) data were utilized in single‐sample gene set enrichment analysis (ssGSEA) analysis. Transcripts per kilobase million (TPM) data was used for CIBERSORTx and ImmuCellAI analysis. GSE16011 cohort from the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) database was downloaded and used to further validate the risk score model. The P27 cell line was derived from GBM patients in the Department of Neuro‐oncology, Cancer Center, Beijing Tiantan Hospital, and was sought after ethical approval (IRB: KY 2021‐153‐03) and patient consent. The cells were handled according to the protocols approved by the ethical committee of Beijing Tiantan Hospital, Capital Medical University. U87, U251, and LN229 GBM cell lines were donated by Beijing Tiantan Hospital Affiliated to Capital Medical University. The genetic types of U87, U251, and LN229 were as follows: U87, U251, and LN229 were IDH1/2‐wildtype cell lines with CDKN2A/B homozygous deletions. EGFR gene amplification in U251 and LN229 cell lines, but not in U87 cells; phosphatase and tensin homolog (PTEN) gene mutant in U87 and U251 cells; PTEN gene wildtype in LN229 cell line; tumor protein p53 (TP53) gene mutant in LN229 and U251 cells; TP53 gene wildtype in U87 cells.First, the RNA sequencing (RNA‐seq) data, somatic cell datasets, and clinical data for all glioma patients were acquired using The Cancer Genome Atlas (TCGA) database. Next, telomerase reverse transcriptase  and x (i) represent the regression coefficients of the multivariate Cox regression model and the expression of Warburg effect‐related genes, respectively.By Kaplan–Meier (K‐M) survival analysis, the prognostic value of Warburg effect‐related genes for GBM was evaluated, and genes with prognostic significance were used for further analysis. Based on these prognostic genes, we performed univariate COX regression analysis and least absolute shrinkage and selection operator (LASSO) regression (R package “glmnet” and “MASS”) to gain the Warburg effect signature for predicting the prognosis of GBM. Eight genes, G Protein‐Coupled Receptor 68 (GPR68), Mitochondrial Pyruvate Carrier 1 (MPC1), Solute Carrier Family 16 Member 1 (SLC16A1), Signal Transducer And Activator Of Transcription 3 (STAT3), Transketolase Like 1 (TKTL1), Ribonucleotide Reductase Regulatory Subunit M2 (RRM2), Mechanistic Target Of Rapamycin Kinase (MTOR), and Toll Like Receptor 4 (TLR4), were obtained by appropriate λ value (representing the appropriate number of genes in the model) and the least Akaike information criterion (AIC) value  to construct the Warburg effect signature. The expression of the Warburg effect signature in GBM patients was defined as the Warburg effect‐related genes (WRGs) score. The following formula was employed for calculating the WRGs score: WRGs score = 2.5p‐value, hazard ratio, and 95% confidence interval of each parameter.The WRGs score and clinical information from GBM patients were integrated for univariate and multivariate Cox regression model analysis. The forest plot was created with the “forestplot” package of R, showing the 2.6Independent characteristics were chosen by multivariate Cox stepwise regression analysis in the TCGA GBM set to build a nomogram to predict the prognosis of GBM patients at 6, 12, and 24 months. The reliability of nomogram prediction was statistically evaluated using the calibration curve, time‐dependent receiver operating characteristic (ROC) curves, concordance index (C‐index), and Brier Score. The nomogram was finally validated using the aforementioned methods in the CGGA validation set.2.7The gene scores of 29 gene sets associated with immunity were evaluated in each sample using the ssGSEA. By comparing differences between 29 immune‐related pathways, patients were categorized into clusters with high and low immune responses. The tumor purity and TME scores  were analyzed by The Estimation of Stromal and Immune cells in Malignant Tumor tissues using Expression data (ESTIMATE). The heatmap and WRGs score together showed the degree of immune infiltration. The percentages of immune cells were calculated using CIBERSORTx and ImmuCellAI. The role of WRGs score on GBM immunotherapy responses was shown using violin plots to show MSI and TMB in the two groups, respectively.2.8n = 5), respectively.Using data on drug response from the Cancer Therapeutic Response Portal (CTRP) and the Cancer Cell Lines Encyclopedia (CCLE), drugs associated with the Warburg effect signature were investigated using Spearman's correlation analysis. The effect of different concentrations of inhibition of arginase with (S)‐(2‐boronoethyl)‐L‐cysteine  ranging from 0 to 1000 μmol/L on the viability of U87, U251, LN229 GBM, and P27 cell lines for 72 h was studied using CCK‐8 assays. The effect of BEC on glucose uptake and lactate generation was studied between BEC‐treated and untreated GBM cell lines. BEC‐treated referred to BEC treated with 100 μM BEC. U87, U251, LN229, and P27 GBM cell lines were divided into treated‐BEC groups and nontreated‐BEC groups. After 48 h of culture, the culture supernatant was collected. Then, the cell viability of two groups was detected by CCK‐8, respectively. Next, the glucose and lactate contents in the culture supernatant were quantitatively analyzed by a glucose detection kit  and a lactate detection kit , respectively. Finally, glucose uptake and lactate generation were calculated for each unit of cell viability. The glucose consumption and lactate generation experiments were repeated five times  complete media with 10% FBS , 1% penicillin–streptomycin , and 2% prophylactic Plasmocin™ . Once every 3 days, the cells were passaged. Every 2 days, the medium was changed.2.10p‐value of <0.05 were considered statistically significant.The R software (version 4.1.1) and Bioconductor package were utilized for all bioinformatics data analysis. The Graphpad prism was used for laboratory data analysis. Spearman's correlation coefficient |R| > 0.3 and a 33.1Using the TCGA GBM cohort, somatic mutation analysis and CNV were carried out to investigate genetic characteristics. Variant classification, variant type, gene mutation proportion, and single‐nucleotide variant (SNV) were all assessed in the somatic mutation analysis Figure . Of the 3.2p < 0.001)    Figure . We perf) Figure . The ris) Figure . In addi) Figure . However) Figure .3.3p < 0.001), radiotherapy (p = 0.013), chemotherapy (p = 0.011), subtype (p = 0.012), and age (p = 0.037) were significantly related to the prognosis in the GBM cohort of the TCGA. Next, WRGs score (p < 0.001), radiotherapy (p = 0.034), chemotherapy (p = 0.044), and age (p = 0.043) were confirmed to independently influence the prognosis of GBM using multivariate analysis. Therefore, the WRGs score calculated by the TCGA GBM cohort was the independent prognostic indicator with the greatest effect on the prognosis of GBM. Moreover, the WRGs score was identified as an independent prognostic characteristic for GBM in the CGGA cohort (p = 0.011)   Figure  and the ) Figure .3.4p < 0.001)   were included, and the results showed that WRGs score had the highest effect on the prognosis of GBM among these factors Figure . Based o) Figure . The cal) Figure . ROC ana) Figure . The C‐i) Figure . The cal) Figure . The C‐i) Figure . The GSE) Figure . The cal3.5Based on previous studies, we hypothesized that the Warburg effect might influence the prognosis of GBM by interfering with the immune microenvironment. Thus, the ssGSEA, ESTIMATE, CIBERSORTx, and ImmuCellAI were utilized to assess the differences in immune infiltration between two groups. The results of the ssGSEA revealed the correlation between 29 immune‐related components and WRGs score, with immune‐related components being more activated in the high‐WRGs cluster Figure . Based o3.6By statistically comparing the expression of Warburg effect signature related antibodies in low‐ and high‐grade gliomas (HGG) on the Human Protein Atlas website, we discovered that the high expression of Warburg effect signature in HGG was highly expressed in the high‐WRGs group, providing a basis for the treatment of GBM by targeting Warburg effect signature. To identify drugs that were sensitive against Warburg effect signature, drug sensitivity analysis was performed using the CTRP and CCLE databases. The correlation analysis between the dose–response AUC of drugs and the gene expression levels was performed to explore the sensitive drugs of the Warburg effect‐related genes Figure . The res4GBM is a glioma with a poor prognosis, drug resistance, and a high incidence of relapse.In our study, the WRGs score was the factor that most significantly affected the prognosis of GBM, although other factors such as surgery,Our prognostic model showed low levels of MPC1 expression and high levels of STAT3, RRM2, and TLR4 expression, suggesting a poor prognosis for GBM patients, which was in agreement with previous studies.Analysis of immune infiltration revealed that the abundance of immune‐suppressive cells, such as Tregs and neutrophils, was considerably higher in the high‐WRGs group as compared to the low‐WRGs group, indicating that the Warburg effect may be one of the reasons for immunosuppression in GBM. A possible mechanism is that the accumulation of lactate by the Warburg effect inside the cell might damage the cellular compartment and halt metabolic processes. Excess lactate must be transported from the cell into the TME to prevent intracellular acidification, which results in the acidification of TME.Targeting the Warburg effect is currently considered a promising cancer therapy strategy,There were a few limitations to our research. The CGGA validation set for this study was obtained after excluding LGG and IDH mutant GBM following the fourth edition of the WHO CNS in 2016, which did not include newly upgraded GBM patients in the WHO CNS5 in 2021. Secondly, the prognostic model of Warburg effect‐related genes has not yet been used in clinical practice, which requires further validation in prospective studies and clinical trials.In conclusion, a trustworthy Warburg effect‐related genes risk scoring model was identified. This model has a significantly excellent predictive value for the prognosis of GBM patients, suggests their immunotherapy response, and identifies new GBM therapeutic targets. In addition, a promising prognostic nomogram was built using a combination of the WRGs score and clinical characteristics to provide an individual prediction of OS and facilitate the selection of effective treatment strategies in the clinic.Rong Zhang: Conceptualization ; data curation ; formal analysis ; investigation ; methodology ; visualization ; writing – original draft ; writing – review and editing . Can Wang: Conceptualization ; data curation ; formal analysis ; methodology ; software ; validation ; visualization ; writing – review and editing . Xiaohong Zheng: Formal analysis ; investigation ; writing – review and editing . Shenglan Li: Data curation ; project administration ; software . Weichunbai Zhang: Data curation ; software . Zhuang Kang: Data curation . Shuo Yin: Data curation . Jinyi Chen: Data curation . Feng Chen: Data curation ; methodology ; supervision . Wenbin Li: Conceptualization ; data curation ; funding acquisition ; methodology ; project administration ; supervision ; writing – review and editing .This study received financial support from the Beijing Advanced Innovation Center for Big Data‐based Precision Medicine, Capital Medical University, Beijing, 100,069, China (2021YFF0901404), the National Natural Science Foundation of China (No.81972338), National Science and Technology Major Project of China (No.2016ZX09101017), Clinical Major Specialty Projects of Beijing, and the Talent Introduction Foundation of Tiantan Hospital (RCYJ‐2020‐2025‐LWB).The authors declare no conflicts of interest.This study was approved by the Beijing Tiantan Hospital of Capital Medical University, Beijing, China. The P27 cell line from passage 27 of primary GBM cells was sought after ethical approval and patient consent.KY 2021‐153‐03.Approval of the research protocol by an Institutional Reviewer Board: N/A.Animal Studies: N/A.Written informed consent was obtained from all the patients before inclusion in the study.Figure S1:Click here for additional data file.Figure S2:Click here for additional data file.Figure S3:Click here for additional data file.Figure S4:Click here for additional data file.Figure S5:Click here for additional data file.Table S1:Click here for additional data file.


To describe clinical and biological features and the outcomes of patients admitted for histoplasmosis in two intensive care units (ICU) in French Guyana and in the French West Indies (Guadeloupe).All patients admitted to these two ICUs for culture-proven histoplasmosis between January 2014 to August 2022 were included in the study. Using univariate and multivariate analysis, we assessed risk factors at ICU admission that were associated with death.Forty patients were included (65% men). Median age was 56 years and simplified acute physiologic score (SAPS) II was 65. HIV was found in 58%, another immunodeficiency was identified in 28%, and no underlying immunodeficiency could be identified in 14% of patients. Within the first 24 h of ICU admission, 85% of patients had acute respiratory failure, 78% had shock, 30% had coma, and 48% had hemophagocytic lymphohistiocytosis. Mechanical ventilation was instituted in 78% of patients and renal replacement therapy in 55%. The 30-day mortality was 53%. By multivariate analysis, factors independently associated with 30-day mortality were SOFA score , time between symptom onset and treatment per day , and hemophagocytic lymphohistiocytosis .Histoplasmosis requiring ICU admission is a protean disease with multiple and severe organ involvement. Immunodeficiency is found in most patients. The prognosis remains severe despite appropriate treatment and is worsened by late treatment initiation.The online version contains supplementary material available at 10.1186/s13613-023-01128-7. Histoplasma capsulatum var. capsulatum is a dimorphic fungus endemic in South America, the Caribbean area, and North America [Histoplasma spp. are highly prevalent. In the French West Indies and French Guiana, up to 30% of the population is thought to have been exposed to Histoplasma capsulatum [ America , 2 Histopsulatum . Histoplpsulatum , even thpsulatum . During psulatum , histoplpsulatum . Accordipsulatum –10, histpsulatum   over an 8-year period. In addition, we compared the characteristics of patients according to the underlying cause of immunosuppression. Finally, we aimed to identify factors associated with 30-day mortality.This retrospective bicentric study was conducted in two ICUs, the Guadeloupe University Hospital  and the Cayenne Hospital  from January 2014 to August 2022. The study was approved by the local ethics committee, which waived consent for retrospective anonymous data collection according to French Law, and it was registered under the number 2223746 v 0. This study follows the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement.Cases were identified through computerized medical records and laboratory microbiology databases of each institution. All consecutive adult patients (> 18 years old) admitted to the ICU for proven histoplasmosis were screened for inclusion.Histoplasma capsulatum by Gomori methenamine silver stain culture [Criteria for histoplasmosis were  symptoms culture  or a pos culture –10.Age, SOFA score, geographic origin, and sex were collected at ICU admission. The underlying condition favoring histoplasmosis was classified as follows:  HIV-infeClinical characteristics within the first 24 h of ICU admission were collected as follows: body temperature, neurological symptoms , gastrointestinal symptoms  and respiratory symptoms .Microscopic examination from broncho-alveolar lavage (BAL), Bone marrow aspirate, and blood were collected. Bacterial and viral concomitant infections diagnosed at ICU admission with biological fluid analysis and cultures  were also collected.All radiological data  were collected and reviewed. When thoracic CT was performed, we reported the presence of macro-nodules, adenopathy, and miliary pattern  pulmonary nodules ).Severe organ involvement within the first 24 h of ICU admission was defined as follows:Coma was defined as a Glasgow Coma Scale score < 8.2 > 92% with respiratory rate > 30/min.Acute respiratory failure (ARF) was defined by an oxygen flow ≥ 5 L/min to maintain SpOShock was defined by mean arterial pressure below 65 mm Hg associated with a lactatemia of > 2 mmol/L and need for vasopressors .Hemophagocytic lymphohistiocytosis (HLH) was defined according to the Saint Antoine criteria .Histoplasma spp. was isolated only from bronchoalveolar lavage with no other sign of extrapulmonary infection.-Histoplasmosis was pulmonary when Histoplasma spp. was isolated from at least another site than lung, with or without lung involvement.-Histoplasmosis was disseminated when Histoplasmosis was classified according to the EORTC and ATS criteria –10:-HistAdvanced life support therapy taken during the ICU stay , the naAnalyses were performed using R (version 4.0.4) . ContinuHistoplasma capsulatum identification was from bronchoalveolar lavage , 86 patients were admitted for histoplasmosis in the two centers. Among them, 40 were admitted in the two participating ICUs: 13 in French Guyana and 27 in Guadeloupe. Histoplasmosis was microbiologically proven in all patients. Table 4–2022, 8Table n = 26, 65%) with a median age of 56 years (44–62), and SOFA was 11 [n = 23, 58%), among whom 30% (n = 7/23) were receiving highly active antiretroviral therapy (HAART). All HIV patients had a CD4 cell count < 100/mm3. Another immunodeficiency was identified in 11 patients (28%), steroid treatment in 23% (n = 9), azathioprine in 3% (n = 1), SOT in 13% (n = 5) and hematological malignancy in 8% (n = 3). Of note, all SOT patients were kidney recipients and were transplanted for three years in median. No underlying immunodeficiency could be identified in the remaining 6 patients (15%); notably, all but 1 had diabetes.Patients were mostly men (A was 11 –15 at ICn = 38), and most patients had pulmonary abnormalities on CT scan when performed , macro-nodules , and abnormal lymph nodes . Neurological manifestations consisted in altered state of consciousness , encephalopathy , and focal deficit . Cerebral CT was performed in 40% of the patients (n = 14) and cerebrospinal fluid analysis in 3 patients and were both normal in all of them. Due to thrombopenia, lumbar puncture was not performed in 9 patients. Brain MRI was performed in 2 patients, without pathological findings. Gastrointestinal involvement was described in 58% (n = 23) of the patients. Symptoms were aspecific, mostly diarrhea  and abdominal pain . Four patients had colitis features on CT. No specific cutaneous manifestations were reported.Duration of symptoms before ICU admission was 23 days –30 and 4n = 8) of the patients , shock in 78% (n = 31), and coma in 30% (n = 12). In patients with shock during the 24 h following ICU admission, norepinephrine posology was 1.0 µg/kg/min (0.3–1.5). Hemophagocytosis lymphohistiocytosis was diagnosed in 19 patients (48%)  had disseminated histoplasmosis, and 20% had pulmonary histoplasmosis.Within 24 h following ICU admission, acute respiratory failure was reported in 85% of the patients  were mechanically ventilated, with a P/F ratio of 110 mm Hg (75–200) at MV onset. Renal replacement therapy was initiated in 55% of patients (n = 22). Among survivors, all were weaned from renal replacement therapy at ICU discharge.Of the patients, 78% (p < 0.001) and a longer time from symptom onset to treatment (p = 0.001) than that observed in HIV-infected patients and in those with another immunodeficiency.HLH was more frequent in HIV-positive patients than in the two other groups. Patients without identified underlying immunodeficiency were characterized by a lower prevalence of disseminated histoplasmosis (n = 34). Liposomal amphotericin B (dose 5 mg/kg/day) was used in 80% of the patients (n = 32) and itraconazole in 20% (n = 8). All survivors received a 14-day treatment course with a switch to itraconazole as maintenance therapy. Time from symptom onset to treatment was 23 days (15–40), and time from ICU admission to treatment was 1 day (0–2). Of note, no patients had antifungal prophylaxis before hospital admission. Once the diagnosis of histoplasmosis was confirmed, all patients received steroids (40 mg/day of prednisone).Antifungal therapy was given before the diagnosis of histoplasmosis to 15% of patients (n = 6) and after the diagnosis to 85% (n = 21). Table p = 0.01), time between symptoms onset and treatment, per day , and HLH  . In our study, the prognosis was grim, being fatal in 53% of patients, and was influenced by illness severity and the time between symptom onset and treatment. Interestingly, delay in treatment initiation was greater among patients without underlying immunodeficiency , but 15% had no identified immunodeficiency. In a recent North American epidemiologic study on predisposing factors for histoplasmosis , 25, immSome specific characteristics of histoplasmosis should be highlighted to help physicians in the diagnosis process. In our series, thoracic CT showed a predominantly miliary pattern. The diagnostic yield of BAL analysis Table  was highn = 22) of affected patients required RRT.Acute kidney injury (AKI) is frequently associated with histoplasmosis (in up to 59% of patients ). The etn = 4). This was probably secondary to the long time between symptom onset and treatment initiation for this sub-population Considering the severity of histoplasmosis in the ICU and the impact of delay in treatment on mortality, this diagnosis should be systematically raised in endemic areas in case of lung miliary lesions, micronodules, or an excavated lesion. In these areas, we suggest adding systematically empirical antifungal treatment in case of pulmonary miliary lesions with shock , and patients without any identified immunodeficiency who had their own contributing characteristics. No specific analysis of factors associated with 30-day mortality in each patient subgroup could be performed, due to the subgroups’ sizes. Third, due to the retrospective study design, selection bias could have occurred, and we were not able to estimate the prevalence of severe histoplasmosis in the two endemic areas. Fourth, as it is informed by patients, time from symptoms onset to hospitalization might be unprecise. However, historical series of histoplasmosis cases were also small in size due to the rarity of the disease. Due to the multicentric nature of our study in two large endemic areas, we think that our data may be consistent with the characteristics of histoplasmosis patients treated in the ICU.In endemic areas, severe histoplasmosis is not uncommon in the ICU. Critically ill patients with histoplasmosis are mostly immunocompromised, with disseminated forms, leading to multiorgan failure. Patients with no underlying immunodeficiency are also at risk. The overall prognosis seems grim, even with appropriate antifungal therapy. Multiple organ failure, hemophagocytic lymphohistiocytosis, and delay of treatment initiation are the main identified risk factors for death.Additional file 1: Table S1. Concomitant infection at ICU admission. Table S2. Clinical and Biological features of patients with Hemophagocytosis lymphohistiocytosis at ICU admission. Table S3. Factors associated with 30-day mortality by univariate and multivariate analysis after excluding patients with no identified immunodeficiency.