Non‐communicable diseases (NCDs) are an important driver of morbidity among ageing people living with HIV (PLWH). We examined the composite role of age and HIV status on NCDs in people living with and without HIV.2), elevated blood pressure (BP) (any systolic BP >139 mmHg or diastolic BP >89 mmHg), obesity (body mass index >30 kg/m2), diabetes mellitus (DM) (fasting glucose ≥126 mg/dl or antidiabetic medication) and dysglycemia (fasting glucose ≥99 mg/dl or non‐fasting ≥199 mg/dl). Multivariable logistic regression with generalized estimating equations was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for factors associated with each NCD. The main exposure of interest was a composite of HIV status and age dichotomized around 50 years. All models were adjusted for study site and sex. The renal insufficiency model was additionally adjusted for elevated BP and dysglycemia.The African Cohort Study (AFRICOS) prospectively enrols participants aged ≥15 years with and without HIV at 12 sites in Kenya, Tanzania, Uganda and Nigeria. From 21 January 2013 to 1 September 2021, we assessed participants for renal insufficiency (estimated glomerular filtration rate <60 ml/minute/1.73 mn = 128 (26.9%), n = 75 (15.8%), n = 8 (1.7%) and n = 40 (8.4%), respectively, for PLWH ≥50. Compared to people without HIV age <50, PLWH age ≥50 had increased adjusted odds of having DM , dysglycemia  and renal insufficiency . There were significant differences by study site, specifically, participants from Nigeria had the highest odds of elevated BP, dysglycemia and renal insufficiency as compared to Uganda.Of 3761 participants with age data, 557 (14.8%) were age ≥50, 2188 (58.2%) were females and 3099 (82.4%) were PLWH. At enrolment, the prevalence of elevated BP, dysglycemia, renal insufficiency and obesity were There was a high burden of NCDs in this African cohort with differences by geographic region. In order to promote healthy ageing with HIV, screening and treatment for common NCDs should be incorporated into routine HIV care with attention paid to geographic heterogeneity to better allocate resources. S22.1AFRICOS is an ongoing prospective cohort enrolling at 12 clinics across five programmes supported by PEPFAR: Kayunga, Uganda; South Rift Valley, Kenya; Kisumu West, Kenya; Mbeya, Tanzania; and Lagos and Abuja, Nigeria . PLWH weThe study was approved by institutional review boards of the Walter Reed Army Institute of Research and all collaborating institutions. All participants provided written informed consent.2.2At enrollment, all participants were administered a medical history, physical exam, demographic questionnaire and underwent phlebotomy. PLWH underwent confirmatory HIV rapid diagnostic testing, CD4 T‐lymphocyte count and HIV Viral Load (VL) . Study v2.33. All data were recorded on paper case report forms and double entered into the ClinPlus platform .Demographic variables, including sex, age, education level, HIV status and clinical care site, are reported for the enrolment visit. For PLWH, ART use and VL stratum were combined into the following categories: not on ART, on ART and VL <1000 copies/ml, and on ART and VL ≥1000 copies/ml. CD4 nadir was categorized as <200, 200–349, 350–499 and ≥500 cells/mm2 calculated using the Modification of Diet in Renal Disease equation [2.Elevated blood pressure (BP) was defined as systolic blood pressure of >139 mmHg, diastolic blood pressure >89 mmHg or receipt of antihypertensive medications. Abnormal BPs were repeated for confirmation. Dysglycemia was defined as fasting glucose ≥99 mg/dl, non‐fasting glucose ≥199 mg/dl or receipt of hypoglycaemic medications. Diabetes mellitus (DM) was defined as fasting glucose ≥126 mg/dl or receipt of hypoglycaemic medications. Renal insufficiency was defined as the estimated glomerular filtration rate <60 ml/minute/1.73 mequation . Obesity2.4The main exposure of interest was a composite of HIV status and age dichotomized around 50 years. Comparisons of demographic characteristics and other parameters across groups of interest were made using Pearson chi‐squared test for categorical variables and the Kruskal–Wallis test for continuous variables. Longitudinal analyses involved multivariable logistic regression with generalized estimating equations, clustered by a participant to account for repeated measures, to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for factors associated with each NCD. NCDs were assessed at every follow‐up visit and the model was time updated. Once diagnosed with an NCD, a participant would not have additional events of that disease. DM and dysglycemia models were adjusted for potential confounders, including study site and sex. Renal insufficiency models were adjusted for elevated BP and dysglycemia given the known risk factors for disease , 17. Ana33.1n = 80, 2.1%). Among PLWH age <50, 1572 (60.0%) had virologic suppression less than 1000 copies/ml. In comparison, a greater proportion of PLWH age ≥50 had virologic suppression less than 1000 copies/ml . Thirty‐six percent (n = 1135) of participants had a CD4 nadir below 200 cells/mm3. Thirty‐five percent (n = 1099) had an enrolment CD4 above 500 cells/mm3. Most PLWH were on a non‐nucleoside reverse transcriptase inhibitor or an integrase strand transfer inhibitor (INSTI); only 5.5% were on a protease inhibitor based regimen.Between 21 January 2013 and 1 September 2021, 3762 participants were enrolled in AFRICOS and 3761 with age data were included in these analyses  of 4.66 (1.74–6.08) years to evaluate factors associated with NCDs. In the multivariable analysis for factors associated with dysglycemia and diabetes Table , there wThe multivariable analysis for renal insufficiency Table  demonstr4NCDs are a leading cause of morbidity and mortality in low‐ and middle‐income countries , 18. In Geographic heterogeneity was seen with NCDs in AFRICOS. Cohort participants in Nigeria had the highest odds of having dysglycemia and renal insufficiency. NCDs account for up to 29% of all deaths in Nigeria . DietaryTreatment of HIV with ART has been implicated in the development of certain NCDs by side effects or toxicity. Long‐term use of tenofovir disoproxil fumarate (TDF) can result in nephrotoxicity. Dolutegravir raises serum creatinine without changing renal function because it inhibits proximal renal tubular secretion of creatinine by organic cation transporters . In addiINSTIs, particularly second‐generation INSTIs, have been implicated in substantial weight gain , 25, 26.This study has multiple strengths, including a diverse population with extensive data collected since 2013. Limitations are that this is an observational cohort with a low number of participants aged≥50, particularly PLWoH in that age group that limit statistical power. Testing for diabetes, dysglycemia and renal insufficiency was not added to study procedures for PLWoH until 2017 and, therefore, these data are not available for these participants. However, data were included for subsequent visits by these participants after amendment implementation. We attempted to adjust for confounders, including study site, age, HIV status, sex and other diseases, known to increase the risk of NCDs; however, we did not adjust for ART exposure differences given the inclusion of PLWoH. We were unable to adjust for other confounders, such as physical activity and nutrition, as these were not collected as part of the study.5This study provides NCD prevalence and characteristics in an ageing sub‐Saharan African PLWH population. There was a large burden of NCDs in this cohort that varied by geographic region. If left unmitigated, NCDs can lead to downstream effects, such as neurologic and cardiovascular disease, causing significant morbidity and mortality. Treating individuals with multiple comorbidities is complex and models of care will need to be developed to appropriately manage these individuals. The geographic component is important because in areas with high burdens of NCDs it will be important to appropriately allocate resources to promote healthy ageing with HIV.The authors have no competing interests to disclose.CCG, JAA, JSC, ALE and DC conceived of the presented research idea. EB, MI, HK, JO, JM and VS carried out the data collection, laboratory activities and reviewed the collected data for quality and reliability. ALE designed the model and analysed the data. ND verified underlying data. DC, ALE, JAA, TAC, NFD, CSP, JSC and CCG contributed to the interpretation of the results. ALE and DC took the lead in writing the manuscript. CCG, CSP and JAA were in charge of overall direction and planning. All authors provided critical feedback and helped shape the research, analysis and manuscript. All authors approve of the final submitted manuscript.This work was supported by the President's Emergency Plan for AIDS Relief via a cooperative agreement between the Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. and the U.S. Department of Defense.The views expressed are those of the authors and should not be construed to represent the positions of the US Army, the Department of Defense or the Department of State. The investigators have adhered to the policies for the protection of human subjects as prescribed in Army Regulation 70‐25.


The pathological phenotype of perineural invasion (PNI) in squamous cell carcinoma (ESCC) is prevalent but highly heterogeneous.Postoperative specimens from all patients with ESCC at Shaanxi Provincial People’s Hospital were evaluated for PNI using haematoxylin and eosin (H&E) staining and S100 immunohistochemistry (IHC). We determined the correlation between PNI status and clinical outcomes.P < 0.05). Multivariate analysis showed that PNI (P = 0.001) was an independent factor affecting disease-free survival (DFS) in ESCC, and a similar result was found for overall survival (OS) (P = 0.017). Further analysis revealed that PNI status was a prognostic factor of DFS (P < 0.001) and OS (P = 0.003) exclusively in pN-negative patients. We also found that patients with the PNI-a subtype had better DFS (P = 0.002) and OS (P = 0.002) than patients with the other three subtypes .Among 349 ESCC cases, PNI was identified in 127 patients (36.3%), and four subtypes of PNI were identified in our study. Correlation analysis confirmed that PNI was related to tumour invasion depth (pT stage) and lymph node status (pN stage) (The pathological phenotypes of PNI are diverse, and the identification of PNI subtypes has important clinical guiding value. Esophageal cancer is the sixth leading cause of death due to malignant tumours with aggressive biological behaviour . Its patA retrospective analysis of patients who underwent radical oesophagectomy between January 2014 and December 2018 was conducted  staining and immunohistochemistry (IHC) of PNI sections were reviewed without the knowledge of the pathology report and re-evaluated for PNI by independent attending pathologists  blinded to the patients’ outcomes. IHC was performed using S-100  monoclonal antibody to evaluate nerves using an automatic immunostainer and Ultra VISION universal DAB  detection kit . The nerve fibre could be clearly identified in the serial section by positive S100 IHC staining. Appendix tissue sections were used as the positive and negative controls. All microscopic analyses were performed using an optical microscope . The diagnostic criteria , 15 for P value of < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS 22.0 and Stata 15.0.The relationship between PNI and clinicopathological characteristics was tested using the chi-square test. The Kaplan–Meier method was used to calculate disease-free survival (DFS) and overall survival (OS), and the difference in survival rates between the groups was tested using the log-rank test. All statistically significant prognostic factors identified in the univariate analysis were included in the multivariate Cox regression analysis, and stepwise regression and collinearity tests were used in our analysis. The chi-square test was used to assess the fit of the Cox proportional hazards model. A two-tailed test with a Overall, 349 patients with ESCC, including 255 men and 94 women, were included in this study. Regarding tumour location, patients with mid-thoracic esophageal cancer were the most prevalent . All patients were updated according to the eighth edition of the TNM staging criteria. In total, 44, 145, and 147 patients were diagnosed with stage I, II, and III disease, respectively. Notably, 15 patients were upgraded from stage III to stage IV by the new staging system and were also included in the analysis (Table P = 0.010). We also found that PNI was associated with lymph node metastasis (pN stage). The incidence of PNI in patients with positive lymph nodes was as high as 48.1% compared with 26.5% in patients with negative lymph nodes (P < 0.001).The overall prevalence of PNI among ESCC cases in our study was 36.3% 127/349). Four types of PNI  were identified: 35 cases of PNI-a, 32 cases of PNI-b, 36 cases of PNI-c, and 24 cases of PNI-d. Next, we analysed the relationship between PNI and the clinicopathological characteristics of patients with ESCC; the details are listed in Table 27/349. FP < 0.001; Table P = 0.037), tumour length (P = 0.002), pT stage (P = 0.001), pN stage (P < 0.001), and pTNM stage (P < 0.001) were all important variables that affected the DFS of patients with ESCC. We included the five variables of sex, tumour length, pT stage, pN stage and PNI identified above in a multivariate Cox model for multivariate analysis  and pN stage (P < 0.001) were independent factors affecting DFS in ESCC. Further analysis showed that in patients with ESCC, the presence of PNI increased the risk of recurrence and metastasis by 1.6-fold  , tumour length (P = 0.013), pT stage (P = 0.002), pN stage (P < 0.001), and pTNM stage (P < 0.001) can serve as predictors of OS in ESCC  and pN  were independent factors predictive of OS in ESCC  and OS (P = 0.002) than patients with the other three subtypes .Moreover, we observed a correlation between PNI phenotypes and clinicopathological characteristics Table . We alsoP < 0.001) and OS (P = 0.003). However, for pN-positive patients, different PNI statuses did not affect DFS (P = 0.615) or OS (P = 0.731) (Fig. Next, we analysed the predictive role of PNI in the prognosis of patients with different pN stages of ESCC. For pN-negative patients, PNI was an influencing prognostic factor for DFS ( P = 0.61 or OS (PThe mode of microinvasion of malignant tumours mainly involves VI and LI. PNI is an under recognized but very important tumour invasion route. Research on the pathological characteristics and prognostic value of PNI has been reported in a variety of tumours, the most common being pancreatic cancer and prostate cancer –18. Whet13. Xu et al. [The incidence of PNI in ESCC varies from 7.9 to 50.7% give the lack of standard diagnostic criteria , 20, 21.The PNI status of ESCC should be evaluated in clinical practice. Although PNI was considered to be an independent prognostic factor of ESCC in some studies , 23, OchThis study had certain limitations, which mainly include the following three aspects. First, the assessment of PNI before and after neoadjuvant therapy was not conducted in this study. Second, single-centre research may lead to bias in the sample selection. Third, although we used H&E and anti-S100 IHC to evaluate PNI, there are currently no definite diagnostic criteria for PNI. These issues require further investigation.In summary, PNI exhibited a nonnegligible invasion pattern in early-stage ESCC. PNI may serve as a potential supplement to the pathological TNM staging of ESCC and may serve as a prognostic predictor of ESCC.


Alpinia oxyphylla Miquel seed has been used to treat gout and hyperuricemia-related symptoms by enhancing kidney functions. Allopurinol is the most commonly used drug to treat hyperuricemia; however, the drug has many adverse effects. Combining allopurinol with another compound could reduce the need for high doses and result in improved safety. We investigated the possible synergistic effects of Alpinia oxyphylla seed extract (AE) and allopurinol in decreasing urate concentrations in rats with potassium oxonate-induced hyperuricemia. This study evaluated the effects of allopurinol combined with AE on levels of serum urate, blood urea nitrogen (BUN), and creatinine in a hyperuricemic rat model. The effects of allopurinol plus AE on xanthine oxidase (XOD) activity and urate uptake were measured. The concomitant administration of allopurinol and AE normalized serum urate and reduced BUN and creatinine. The attenuation of hyperuricemia-induced impaired kidney function was related to downregulation of renal urate transporter 1 and upregulation of renal organic anion transporter 1, with inhibition of serum and hepatic XOD activities. The antihyperuricemic effects of allopurinol were enhanced when combined with AE. These results suggested that the combined use of allopurinol and AE may have clinical efficacy in treating hyperuricemia.In traditional medicine,  Excess urate in the blood is an important risk factor for the development of gout, diabetes, and kidney disease –3. HyperThe biosynthesis of urate in the liver is mediated via xanthine oxidase (XOD) in two stages (conversion of hypoxanthine to xanthine and oxidation of xanthine to urate) of urate generation . ReabsorAllopurinol is a common urate-lowering medication used to treat gout and hyperuricemia . As a puAlpinia oxyphylla Miquel (Zingiberaceae family) plant is a well-known traditional herbal medicine in Asia. The dried seed is used to treat diuresis, enuresis, intestinal disorders, and chronic glomerulonephritis [A. oxyphylla extract and its constituent nootkatone reduced serum urate concentrations in a potassium oxonate- (PO-) induced hyperuricemia rat by enhancing urate excretion [A. oxyphylla regulated URAT1 and OAT1, the urate transporters responsible for the reabsorption and secretion of urate in the kidneys. Combining A. oxyphylla with the XOD inhibitor, allopurinol, may provide a dual mechanism for lowering serum uric acid concentrations via increased urate excretion and reduced urate production. Furthermore, we determined whether A. oxyphylla potentiated the antihyperuricemic effects of allopurinol in a hyperuricemic rat model induced by PO.The ephritis . In tradephritis –15. The ephritis , 17. We A. oxyphylla seed extract was provided by Kwangdong Pharm Co. Ltd. , and a voucher specimen (No. P008) was stored at the Korea Institute of Oriental Medicine (KIOM), Daejeon, Republic of Korea. The plant was authenticated based on the macroscopic characteristics provided by the Classification and Identification Committee of KIOM. A preliminary investigation of various extracts  of A. oxyphylla seeds showed that a 30% ethanolic extract had the most antihyperuricemic activity [A. oxyphylla (250 kg) seed was extracted with 30% ethanol solution for 9 h at 75°C. After filtration, the residue was reextracted with an aqueous solution for 9 h at 95°C. After filtration, the combined filtrates were concentrated under reduced pressure and sterilized at 95°C for 1 h. Allopurinol was obtained from Sigma-Aldrich . Allopurinol and A. oxyphylla extract were administrated at a 1 : 200 ratio (w/w) for the combination treatment. In our previous study, the concentration of the main component of A. oxyphylla seed extract, nootkatone (1.03 ± 0.13 mg/g), was determined by UPLC analysis for standardization [activity . Thus, tdization .ad libitum access to food and water. The protocol was approved by the Committee on Animal Care of the KIOM, and animal procedures were permitted in accordance with the Guide for the Care and Use of Laboratory Animals .Male Sprague Dawley rats  were obtained from Orient Bio . They were adapted in an air-conditioned animal room at a temperature of 22 ± 1°C and humidity of 50 ± 10%. The rats had n = 5 each): the normal control group (NC), the PO-treated hyperuricemia group, the PO+1 mg/kg allopurinol group (AP), the PO+200 mg/kg AE group, and the PO + combination of 1 mg/kg AP and 200 mg/kg AE group . The doses of AP and AE used in these experiments were determined from preliminary dose-range experiments . Urine samples were collected 2 h later using a metabolic cage following sample administration. Urate levels from serum and urine were determined using a uric acid assay kit . Serum interleukin (IL)-1β levels were determined using an ELISA assay kit . Glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), creatinine, and blood urea nitrogen (BUN) in the serum were measured using a biochemical analyzer . Fractional renal clearance of urate  was calculated by the formula FCU, renal clearance of urate/renal clearance of creatinine = (urine urate × serum creatinine)/(serum urate × urine creatinine) × 100.Blood from rats was collected 2 h following AP, AE, or AP + AE administration, and the serum was separated by centrifugation  according to the manufacturer's protocols. Protein concentrations were measured using a DC protein assay  using bovine serum albumin as the standard to normalize XOD activity. Liver XOD activity was presented as nanomoles of urate formed/min/mg of protein.μg) were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After transfer, the membranes were blocked with EzBlock blocking buffer  for 1 h and incubated with URAT1 , β-actin, GLUT9, OAT1, OAT3 , and IL-1β (Abcam) antibodies at 4°C overnight. The protein bands were visualized using an ECL detection reagent  using ImageQuant LAS 4000 . The band densities from the obtained images were determined by densitometry using Image J1.49 software . Target protein levels were normalized to β-actin.Protein extracts from kidney tissues were homogenized with PRO-PREP protein extraction solution  and centrifuged . Protein concentrations were measured using a DC protein assay (Bio-Rad). The proteins . Significant differences of the results were tested by one-way analysis of variance followed by Tukey's multiple comparison test using Prism 7.0 software . Statistical significance was considered as Previously, to identify the most effective combination among the combinations of allopurinol and AE, experimental preparations were made by combining allopurinol and AE at different proportions of 1 : 100, 2 : 100, and 1 : 200. The combination of allopurinol and AE at a 1 : 200 ratio was the most effective in decreasing serum uric acid levels among the three mixtures tested. Thus, we selected the allopurinol and AE combination at a ratio of 1 : 200 for further studies.p < 0.001), indicating effective establishment of the hyperuricemia rat model. Compared with the PO group, serum urate concentrations were not reduced or reduced slightly after treatment with allopurinol (1 mg/kg) or AE (200 mg/kg) alone. However, when a combination of allopurinol and AE (1 : 200) was given, serum urate concentrations were significantly decreased . Next, to evaluate the effects of the combination on urate excretion, urine urate levels were determined. Urine urate levels and renal urate excretion as fractional renal clearance of urate were significantly increased by the combination of allopurinol and AE (p < 0.05), indicating that the combination of allopurinol and AE increased kidney urate excretion, resulting in decreased serum urate levels in PO-induced hyperuricemia rats.The PO group had significantly higher serum concentrations of urate, when compared with the NC group (p < 0.05). AP or AE alone did not affect either serum BUN or creatinine, but the AP and AE combination significantly reduced these levels . These data indicated that the AP and AE combination decreased PO-induced liver and renal dysfunction.We evaluated serum levels of GOT, GPT, BUN, and creatinine in PO rats. GOT and GPT concentrations did not differ significantly between the other groups; although, GOT levels were decreased by treatment with the combination, when compared with AP alone Figures . The PO p < 0.001 compared with the PO group), decreasing them even lower than in the NC group. AE alone showed a minor effect on serum and hepatic XOD levels. Treatment with the combination significantly suppressed these levels (p < 0.001 compared with the PO group) and enhanced the activities of AE on serum and hepatic XOD levels . These results suggested that the enhanced effects in serum uric acid levels observed after treatment with the combination of AP and AE were related to changes in serum and hepatic XOD activities.AP and the combination of AP and AE had significant inhibitory effects on serum and hepatic XOD levels of rats (p < 0.05). Treatment with the combination of AP and AE increased OAT1, when compared to the PO group (p < 0.05), and increased these changes, when compared with AP or AE alone . AE alone and in combination with APl decreased URAT1 protein levels (p < 0.05 compared to the PO group). These results suggested that the enhanced effects of the combination treatment were related to changes in renal transporter expression.The effects of the combination of AP and AE on OAT1, OAT3, URAT1, and GLUT9 protein levels in the kidneys of hyperuricemic rats are shown in β protein in the serum and kidney . AP or AE alone had only minor effects on serum IL-1β levels, but treatment with the combination led to a significant decrease (p < 0.05). In the kidney, AE alone and combined with AP decreased IL-1β levels, when compared with the PO-treated group (p < 0.01). Mild renal tubular dilatation, dilation of Bowman's space, hemorrhage, tubular vacuolar degeneration, and inflammatory cell infiltration were observed in the kidneys of PO-induced hyperuricemic rats  and inflammation without any side effects in animal model studies . The resAP is better known for the management of hyperuricemia. The rationale for use of AP in hereditary renal hypouricemia patients was to decrease the generation of UA, thus, decreasing the filtered UA load and lowering the risk of precipitation of UA in tubules. With this approach, it was reported that use of AP effectively prevented recurrence of renal injury in this patient . Thus, uIt was also reported that common dysfunctional variants of the high capacity ATP-binding cassette urate transporter (ABCG) gene were major risk factors of gout and hyperuricemia, involving a decrease in urate excretion . In a prThe active metabolite of AP, oxypurinol, is a substrate of URAT1. AE may inhibit the uptake (tubular reabsorption) of oxypurinol through URAT1 after its excretion in urine, thus reducing the blood exposure of oxypurinol. However, further study is needed to evaluate the potential pharmacodynamic drug-drug-interactions between the selective urate excretion inhibitor, AE, and the XO inhibitor, AP .Our results suggest that there may be medicinal value in combining low dose AP with AE for the treatment of hyperuricemia, especially in patients with renal insufficiency. The commonly known dose-related side effects of AP could be attenuated with the addition of AE. However, safety and long-term studies of the combination for clinical use should be further investigated.A. oxyphylla potentiated the effect of AP on serum uric acid concentrations in a hyperuricemic rat model induced by PO. Combination treatment of AP and AE provided a dual mechanism for lowering serum uric acid concentrations, involving increased excretion via regulation of URAT1 and OAT1 and reduced urate production via XOD inhibition. These results suggest that the combination of AP and AE may have clinical value for treating hyperuricemia.We determined whether


Spinal cord injury (SCI) is a debilitating condition for which no definitive treatment has yet been identified. Notably, it influences other tissues through inflammatory reactions and metabolic disturbances. Therefore, fingolimod (FTY-720), as an FDA-approved inflammatory modulator, would be promising. In the present study, nanocarriers with two distinct monodisperse particle sizes of 60 (nF60) and 190 (nF190) nm were prepared via low-(stirring) and high-energy (probe ultrasound) emulsion oil in water (O/W) methods. Larger nanocarriers showed higher EE% and sustained-release profile than smaller nanocarriers. Neural stem cell (NSC) viability and lactate dehydrogenase (LDH) release were studied in the presence of nanocarriers and free FTY-720. The results indicated that nanocarriers and free FTY-720 enhanced NSC viability compared with the control group. However, nF190 induced significantly less cell membrane damage than nF60. Nanocarriers and free FTY-720 enhanced motor neuron recovery in SCI rats, while body weight and return to bladder reflux by nF190 were significantly higher than those in the nF60 group. Return to bladder reflux might be due to the role of FTY-720 in the regulation of detrusor muscle tone and preservation of the integrity of vessels by acting on endothelial cells. Moreover, nF190 gained higher soleus muscle weight than the free drugs; probably decreasing proinflammatory cytokines in the soleus diminishes muscular atrophy in SCI rats. In summary, it might be said that larger nanocarriers with sustained-release profile and less cell membrane damage seem to be more efficient than smaller ones to manage SCI and enhance bladder reflux. These data will help pharmaceutical companies select the correct particle size for nanodrugs and develop more efficient drug formulations to treat SCI. Spinal cord injury (SCI) is a severe and debilitating condition that leads to a variety of complications, from transient neurologic impairment to permanent motor and sensory function loss . AccordiκB translocation [Methylprednisolone, a synthetic anti-inflammatory and immunosuppressive glucocorticoid, is the current gold standard for SCI patients. However, the safety and efficacy of this therapeutic agent have been subject to debate . We proplocation . Howeverlocation .Medical nanotechnology strategies have provided significant breakthroughs over the past several decades. Nanoparticle formulations have been successfully employed to treat and ameliorate many diseases and disorders, including advanced cancer, diabetes, HIV, and cardiovascular disease . A largeDespite several investigations on the potential of nanocarriers for SCI recovery, we do not know which particle size would be effective in recovering spinal cord injury. These data will help pharmaceutical companies develop more efficient drug formulations to treat SCI. The present study responds to whether FTY-720 nanoemulsions enhance therapeutic efficacy compared to their bulk counterparts in a contusion SCI rat model. Meanwhile, it defines which particle size is more efficient for SCI recovery.w/w) mineral oil, 6% (w/w) Span 80-Tween 80 (surfactant), 1% (w/w) ethanol (cosurfactant), and distilled water was exposed to either a magnetic stirrer  and/or an ultrasound probe  to obtain 190 nm and 65 nm oil-in-water (o/w) fingolimod nanoemulsions. Nanoemulsions were then stored in sealed glass vials covered with aluminum foil for further investigation. In this paper, NF60 and NF190 will be referred to as fingolimod nanoemulsions with 65 nm and 190 nm particle sizes, respectively.FTY720 nanocarriers at two different particle sizes with equal concentrations of FTY-720, oil, surfactant, and cosurfactant were prepared via low (stirring) and high-energy (probe ultrasound) emulsion oil in water (O/W) methods . In brieThe hydrodynamic particle size and zeta potential of nanocarriers were studied using a dynamic light scattering (DLS) apparatus  at a refractive index of 1.531 and neutral pH to avoid the impact of the false charge on the zeta potential. The assay was performed in triplicate, and the mean ± SD was reported.The surface morphology, shape, and size of the nanoparticles were evaluated using SEM . Before imaging, one droplet of nanocarrier was mounted on a glass slide and dried. Then, the samples were coated with gold, and SEM was recorded at 20 kV.Ultrafiltration is a gold method to evaluate nanocarriers' entrapment efficacy (EE). Indirect method was used to evaluate the amount of encapsulated drug. The newly synthesized nanocarriers in Amicon microtubes were centrifuged for 15 min at 14000 G. The supernatant that contained the unencapsulated drug was removed, and absorbance was read using RIGOL ULTRA 3660 spectrophotometer at a wavelength of 217 nm. EE was calculated using the following formula;C is the total amount of FTY-720, and TC is the free amount of FTY-720, which was detected only in the supernatant media. The assay was performed in triplicate, and the values provided are the mean ± SD of three independent experiments.where Franz diffusion cell was used to investigate the release profile of FTY-720 nanocarriers. 1 ml of each nanoemulsion was placed on a dialysis bag at the receptor part of the Franz diffusion cell. Franz diffusion cells was filled with PBS solution. The receptor and sampling port were completely covered with parafilm. Franz diffusion cells were placed on a shaker at 37°C at 100 rpm for 48 hours. For sampling at different times, 1 ml of the solution is pipetted out of the sampling port and immediately replaced with 1 ml PBS, and the tube end is completely closed again with parafilm. Sampling was performed at interval times of 1, 10, 20, 30 min, 1 h, 2 hours, 4 hours, 24 hours, and 48 hours. The absorbance of the samples was read using a RIGOL Ultra 3660 spectrophotometer at a wavelength of 217 nm, and the accumulated release rate of the drug at different times was plotted by Excel.4 cells/well) for 24 h, and then, FTY-720 and FTY-nanocarriers were added to NCSs at a concentration of 10 ng/ml for 48 h. The cell media was removed, and 100 μl of 3--2,5 diphenyltetrazolium bromide  (0.5 mg ml−1 PBS) was added to the triplicate wells at 37°C in 5% CO2 and 95% moisture for 4 h. DMSO was added, and after 20 min, the absorbance was read using an ELISA reader (Bio-Tek) at 570 nm. The assay was repeated three times in triplicate, and the mean ± SD was reported.Neural stem cells (NSCs) were isolated from the subventricular zone (SVZ) of the lateral ventricles and seeded in NPBM as a cell culture medium. To evaluate the cell viability of nanocarriers and bulk drug, an MTT assay was performed. NSCs were seeded in a 96-well plate . After 20 min, the absorbance was read using an ELISA reader (Bio-Tek) at 490 nm. The assay was repeated three times in triplicate, and the mean ± SD was reported.Isolated NSCs were treated with FTY-720, nF190, nF60, and the control group. After 24 h, 100 μl of the nanocarriers, FTY-720, and PBS at a concentration of 10 ng/ml were injected using a 26 gauge Hamilton syringe at a speed of 1 μl/min at the lesion epicenter. The muscles and skin were sutured, and the animals were kept on a heating path until they awoke. Animals were monitored constantly and maintained with free access to water and food. They received antibiotics (5% gentamicin) daily for five days postsurgery.The Iranian Ethical Committee of Iran University of Medical Sciences approved all experimental protocols and animals used in this study (IR.IUMS.REC.1399.087). All procedures were performed in accordance with the Iranian Ethical Committee for Animal Care and use. The study was carried out in compliance with the ARRIVE guidelines. All experimental procedures were performed in accordance with the relevant guidelines and regulations. Acute severe SCI was induced in 24 male Wistar rats (220–250 g) using the weight compression method, with 6 rats in each group . Rats weThe Basso Beattie Bresnahan (BBB) locomotor open-field locomotor test was carried out to examine hind limb stepping movements at days 1, 7, 14, 21, 28, 35, and 42 postinjury. The rat was placed in a Plexiglas circular apparatus  with a nonslip floor. BBB score assessment was performed by a portable camera for 4 min, evaluated by two blinded independent observers, and averaged from both hind limbs. The scores ranged from 0 to complete paralysis and 21 to normal locomotion.After SCI, the sacral micturition center may send signals to the bladder to squeeze. Bladder emptying was performed twice a day manually until reflex function was regained. The regain of spontaneous bladder function was regarded as a measure of recovery. The mean ± SD was reported for the four groups.Weight gain is a marker of recovery in SCI. Animals were weighed before surgery and at days 1, 7, 14, 21, 28, 35, and 42 postinjury. Then, weight gain was compared on the first day. The mean ± SD was reported for the four groups.ndpost surgery. To dissect the soleus muscle, the skin was detached, and the Achilles tendon was removed. After lifting the gastrocnemius muscle, the underlying soleus muscle was exposed, and then both muscles were sectioned for weighing. The mean ± SD was reported for the four groups.The soleus and gastrocnemius muscles are essential muscles in walking and running. Muscle atrophy is adjacent to less movement. Therefore, in this study, the weight of the soleus and gastrocnemius muscles was measured in rats on the 42μm were obtained from the paraffin-embedded spinal cords. Sample slides were stained with hematoxylin and eosin (H&E) and covered with coverslips. The camera captured images at a resolution of 40X.On day 42 postinjury, rats were transcardially perfused with PBS and fixed with 4% paraformaldehyde in 0.1 M PBS. Spinal cords were isolated, and transverse sections with a thickness of 5 p value less than 0.05.GraphPad Instat software version 3 was applied to analyze particle mean size, zeta potential, cell viability, body weight, muscle weight, and BBB scores. Data were reported as the mean ± SD. One-way ANOVA  was used for statistical analysis. The significance level was set at a One of the most challenging issues in pharmaceutics is defining an effective particle size to recover SCI. Recent decades have witnessed an explosive growth in the design and delivery of nanomedicine. In this regard, FTY nanocarriers with two distinct particle sizes were prepared and compared, and their therapeutic efficacy was investigated in an SCI rat model. We assumed that encapsulation of FTY-720 into a nanoemulsion drug delivery system probably promotes neuronal survival and functional reconstruction of this powerful immunomodulatory medicine.p < 0.001). Meanwhile, they showed that the monodisperse nF60 and nF190 nanocarriers had PDI values of 0.1 and 0.2, respectively . Smaller nanocarriers showed less negative ζ-potential than larger nanocarriers. However, both showed ζ-potentials of approximately -10 mV.SEM micrographs revealed that both the nF60 and nF190 nanocarriers had spherical morphology with uniform size and homogeneous distribution without agglomeration Figures . Environp < 0.0002). Larger nanocarriers had higher capacity to entrap the FTY-720.The EE% was measured using the ultrafiltration method. Results showed NF190 had higher EE% (83.42% ± 1.22) than NF60 (65.98% ± 1.82). There was significant difference between the EE% of both nanocarriers (p > 0.05). After 24 hours, the release rate of the two drugs was different (p < 0.001). This difference continued until the second day and at the end of 48 hours, and the release slope of the NF190 was lower than the release slope of the NF60 (p < 0.001). After 48 hours, in the larger sample, the accumulated release rate was 55% and in the smaller sample, and the accumulated release rate was 74% (p < 0.001). Larger nanocarriers that had higher EE% released FTY-720 in slower rate and more sustained-release profile than smaller nanocarriers . However, the control group had significantly less cell viability than the nanocarriers and bulk FTY-720 (p < 0.001). Nevertheless, both nanoemulsions manifested higher cell viability compared to the control group. It seems that FTY-720 can significantly enhance NSC viability and may be considered a promising drug for neurogenesis -M17 cells. These data are contrary to our recent results and are due to different cell types. Therefore, in neurotoxicity investigations, the cell type is critical, and we cannot refer to an outcome to other cell types. Although nanocarriers with a particle size of 60 nm may be considered a candidate particle size in dopaminergic cells, larger nanocarriers of 190 nm will act as the preferred particle size for SCI recovery. In addition, silica NPs in the range of 200 nm exhibit significantly higher neuronal viability than small NPs (50 nm) through calcium perturbation and apoptosis mechanisms . Howeverp < 0.01). At the same time, there was no significant difference between the other groups (p > 0.05). In other words, it seems that larger nanocarriers induced minor NSC membrane damage, while there was no significant difference between the cell membrane damage of smaller nanocarriers and bulk FTY-720 (p > 0.05)  . In summNanocarriers and FTY-720 were directly injected into the lesion to investigate their effects on the SCI model. In vivo SCI models were exerted on Wistar male rats employing the weight drop contusion model. Blunt injury models, including weight drop, represent human injuries and can efficiently study secondary damage . Herein,p < 0.001). There was no significant difference between the motor neuron recovery of nanocarriers and free FTY-720 (p > 0.05). These findings were in accordance with the MTT assay data. It seems that NSC viability has a direct impact on the potential of nanocarriers to enhance motor neuron recovery. To further investigate the effect of nanocarriers on SCI rats, bladder reflux and body and muscle weights were evaluated.To assess motor function recovery, BBB open-field locomotor rating scales were carried out in contusion rat models that received local FTY- nanocarriers and bulk drug for six weeks postinjury . In the p < 0.01), free drug (p < 0.05), and the control group (p < 0.001). There was no significant difference between the return of bladder reflux in the nF60 and free drug groups (p > 0.05). Probably, the more sustain and longer release profile of NF190 resulted in quicker return of bladder reflux. Based on these results, although larger nanocarriers could enhance motor neuron recovery at the nF60 and free drug scores, they positively impacted bladder reflux, which is very important in SCI patients. Lee et al. demonstrated that daily IP injections of FTY-720 for four weeks following a contusion model in a Long-Evans hooded rat model significantly enhanced functional outcomes and bladder recovery [Impaired bladder function is another incapacitating consequence of SCI in humans and animals. The degree of SCI severity is correlated with bladder dysfunction . In otherecovery . Moreoverecovery .p < 0.001). By the time, the body weight of rats treated with nanocarriers was significantly enhanced compared to the control group and free drug at four weeks. nF190 induced significantly higher body weight than nF60 at 42 days posttreatment (p < 0.001).Body weight changes were recorded weekly as an indicator of general health and recovery . The weip < 0.001). Based on these findings, it seems that although there was no significant difference in motor neuron recovery of nanocarriers and free drugs, gained body weight and bladder reflux, as markers of health and recovery in rats, were enhanced using nF190 and nF60. Eventually, the return of the bladder reflex in SCI rats positively impacted body weight and general recovery.Meanwhile, nF60 significantly influenced higher body weight than the free drug (nd day. The results showed no significant difference between the gastrocnemius weights of the groups (p = 0.0982). There is some difference between the two types of gastrocnemius and soleus muscles, in which the gastrocnemius is predominantly glycolytic muscle while the soleus is more largely slow-twitch muscle [p < 0.01) and nF60 (p < 0.05) groups than in the control group. nF190 showed significantly higher soleus weight than the free drug (p < 0.05)  . Probablt period . Howevert period , and theeriod [α , 38, 39,ndday posttreatment. At the same time, the cavity had a smaller size in nF190. Since a severe model was induced in the spinal cord, the astroglial scar could not completely recover in all groups, and fibroconnective tissue was observed in the cavity of all groups. However, the nanocarriers showed less cavity size compared to others. It seems that nanocarriers help the astroglial scar to be reconstructed (Histological evaluation was performed using H&E staining. As demonstrated in structed .Analyzing the functional recovery of SCI rats treated with free FTY-720, nF60, and nF190 indicated that nF190 remarkably improved the outcomes of faster regain of bladder reflux, gain of body weight, and soleus muscle compared to free FTY-720 and in some parts more than smaller nanocarriers. Kong et al. discovered that PLGA microfibers containing FTY-720 and NSCs reduce the glial scar cavity size, suppress astrocyte differentiation, and induce NSC differentiation into neuron oligodendrocytes, which are critical for axonal reconstruction. The in vivo results of that study pointed out the remarkable efficacy of FTY-720 loaded into electrospun fibers on motor recovery in a spinal cord transection rat model . MoreoveThe high-energy method using an ultrasonicator and the low-energy method using a magnetic stirrer were employed to synthesize nanoemulsions with 60 and 190 nm particle sizes, respectively. Particle size was analyzed using SEM and DLS. Nanocarriers were biocompatible and exhibited higher cell viability than the control group. Local delivery of the FTY-720 nanoemulsion after the contusion model in an SCI rat model significantly improved hindlimb motor function recovery. Collectively, our data demonstrated that nF190 provides us with neuroprotective and neuroregenerative properties. This synthetic nanocarrier not only impedes further damage but also helps to improve motor dysfunction and recovery. Nanocarriers at a particle size of 190 nm through higher EE%, sustained-release profile of FTY-720 that releases less amount of FTY-720 during a specific time interval, enhanced cellular uptake positively impact bladder reflex, bodyweight, and muscular weight. Eventually, smaller nanocarriers through enhanced cell membrane damage than nF190 exhibit less beneficial effects than nF60 in SCI rats. The results of the presented investigation not only are important for pharmaceutical companies as a view of neurosurvival but also are valuable to select the favorable particle size in the treatment of spinal cord injury. It might be said that not only body weight and return to bladder reflux by larger nanocarriers was significantly higher than smaller nanocarriers but also larger nanocarriers promoted higher recovery of spinal cord injury. We suggest that nanocarriers can be studied in a moderate SCI model to significantly show the impact of particle size on motor neuron recovery.


The COVID-19 pandemic uniquely affects patients with neurologic and developmental disabilities at the Kennedy Krieger Institute. These patients are at increased risk of co-morbidities, increasing their risk of contracting COVID-19. Disruptions in their home and school routines, and restrictions accessing crucial healthcare services has had a significant impact.A Pandemic Intake questionnaire regarding COVID-19 related medical concerns of guardians of patients was distributed using Qualtrics. Data from May-December 2020 were merged with demographic information of patients from 10 clinics (Center for Autism and Related Disorders (CARD), Neurology, Epigenetics, Neurogenetics, Center for Development and Learning (CDL) Sickle Cell, Spinal Cord, Sturge-Weber syndrome (SWS), Tourette's, and Metabolism). A provider feedback survey was distributed to program directors to assess the effectiveness of this intervention.p < 0.001) and inability to obtain therapies (p < 0.001) and surgeries (p < 0.001). Guardian responses from CARD had increased reports of worsening behavior (p = 0.01). Providers increased availability of in-person and virtual therapies and visits and made referrals for additional care to address this. In a survey of medical providers, five out of six program directors who received the responses to this survey found this questionnaire helpful in caring for their patients.Analysis included responses from 1643 guardians of pediatric patients . Guardians of patients in more medically complicated clinics reported perceived increased risk of COVID-19 (This quality improvement project successfully implemented a pre-visit questionnaire to quickly assess areas of impact of COVID-19 on patients with neurodevelopmental disorders. During the pandemic, results identified several major areas of impact, including patient populations at increased risk for behavioral changes, sleep and/or disruptions of medical care. Most program directors reported improved patient care as a result. The COVID-19 pandemic uniquely impacted the medical care of patients with neurodevelopmental disabilities. These patients are at increased risk for impaired motor function, cognition, and social interaction, making them more susceptible to COVID-19 related complications , 3, whicThe Kennedy Krieger Institute provides outpatient medical services and care for children and adolescents with disorders involving the brain, spinal cord and musculoskeletal systems. The Kennedy Krieger Institute approaches neurodevelopmental disorders through a multi-professional treatment lens, allowing for expansive integration of changes and improvements in the quality of care. The complex, inter-related factors, unique to patients with neurodevelopmental disorders influence the impact of the COVID-19 pandemic. We hypothesized that clinicians providing medical care during the pandemic would be assisted by this information. We aimed to systematically provide essential COVID-19 pandemic related information to clinicians before their visits with the family, and improve providers ability to deliver high quality care .This project included 10 clinical programs, led by seven clinician program leaders, including the Center for Development and Learning (CDL), the Center for Autism and Related Disorders (CARD), Tourette's syndrome clinic, Neurogenetics, Neurology, Metabolism clinic, Epigenetics, Sturge-Weber syndrome clinic, Sickle Cell disease clinic, and the Center for Spinal Cord Injury. The Center for Development and Learning (CDL) sees patients diagnosed with cognitive impairments, anxiety, attention deficit hyperactivity disorder, learning disorders, language delays and social difficulties. The Neurology clinic sees patients diagnosed with brain injury, leukodystrophies, Rett syndrome, stroke and epilepsy. Over 4,000 patients  were seevia web-link to patients prior to their in-person or telehealth clinic visits. The 33 item Pandemic Intake questionnaire collected information regarding the impact that COVID-19 had on their health, medication, and treatment , as the reference groups against which all other groups were compared. Chi-square analysis determined if responses differed between those in more or less medically complicated clinics. A full factorial binary logistic regression (with interaction) determined whether responses differed between group  and time of survey completion (early vs. later pandemic), and identified which factors drove statistical significant associations. The clinics noted as medically more complicated include: Neurogenetics, Neurology, Spinal Cord Injury, Sturge-Weber syndrome, Epigenetics, Metabolism and the Sickle Cell clinic. CARD, CDL and the Tourette's clinic were noted as less medically complicated. In the medically more complicated clinics , patientResponses to the provider feedback survey  = 158.6, p < 0.001). Fifteen percent seen in the Center for Development and Learning reported an increased perceived risk for their children ; those from more medically complicated programs, i.e., Spine (59%), Other (58%), Sturge-Weber Syndrome (49%), Neurology (44%), and Neurogenetics (35%) clinics were 3.29–8.77 times more likely to report a perceived increased risk of contracting COVID-19 (Three hundred and thirty-seven respondents (20.6%) agreed (“yes”) or were “unsure” if their child was at increased risk of contracting COVID-19. 5.7% reported that this was due to asthma/other lung disease and 0.7 reported that was due to heart disease. Out of all respondents, 2.9% reported that their child had asthma/other lung disease and 0.2% reported their child having heart disease at the time of completing the questionnaire. Clinic type was a significant predictor, but sex, age, and race were not  reported a worsening in their child's mood during the pandemic;  = 43.2, p < 0.001). Race and age were significant predictors; sex and clinic type were not. There was a 1.003 cumulative increase of reports in worsened mood with each increase in age by month (p = 0.01). Guardians of Black and African American (14%), Other (14%), and Asian (15%) patients were 0.48–0.53 times less likely to report worsening in mood compared to White respondents (24%) (Twenty percent (ts (24%) .n = 327) of guardians reported worsening in their child's behavior during COVID-19  = 36.8, p = 0.001). Race, age, and clinic type were significant predictors; sex was not. Reports of worsening in behavior during the pandemic was significantly different across race and ethnicity (p = 0.002), type of clinic (p = 0.014), and across age (p = 0.047). Guardians of Black and African American (17%) and Asian (13%) children were 0.667–0.486 times less likely to report worsening in behavior, compared to parents of White children (23%). Twenty percent of guardians of CDL patients reported a worsening of behavior, while guardians of CARD patients (33%) were 2.05 times more likely to report worsening behavior, and respondents from the Spinal Cord clinic (3%) were 0.12 times less likely to report worsening behavior. There was a 0.998 cumulative decrease in reports of worsening behavior with each increase in age by month (Twenty percent (by month .n = 191) indicated the child's sleep worsened during the pandemic  = 24.8, p = 0.037). Clinic type was a significant predictor; guardians of CARD patients (22%) were 2.2 times more likely to report worsening sleep compared to CDL patients (12%). Sex, race and age were not predictors.Regarding sleep, 11.7% (pandemic . The stan = 81) reported their child experienced worsened appetite. The statistical model correctly classified 95% of the sample, χ2  = 28.2, p = 0.013). Clinic type, age and race were significant predictors; sex was not. Guardians of patients seen at the SWS center (9%) were 2.4 times more likely to report worsened appetite then CDL patients (5%). Black and African American patients (8%) were 1.9 times more likely to report worsened appetite than White respondents (4%). There was a 1.006 cumulative increase in reports of worsened appetite with increase in age by month  of guardians reported increased difficulty accessing therapies for their children during the pandemic. The statistical model correctly categorized 80.4% of the sample, χ2  = 121.5, p < 0.001). Race, age, and clinic type were significant predictors, but not sex. Twenty-three percent (n = 200) of guardians of White children indicated increased difficulty accessing therapies; Black and African American respondents (14%) were less likely to report difficulty accessing therapies. Guardians from the Neurogenetics (38%), Other clinics (27%), and the Spinal Cord clinic (72%) were 2.63–12.92 times more likely to report difficulty accessing therapies as compared to 17% (n = 206) of those from CDL. There was a 0.993 cumulative decrease in reports of difficulty accessing therapies with each increase in age by month. Finally, 4% (n = 66) of guardians indicated difficulty accessing surgical services during the pandemic. The statistical model was significant and correctly categorized 96% of patients, χ2  = 43.7, p < 0.001). Clinic type was a significant predictor; age, sex, and race were not. Respondents from the Tourette's (8%), Spinal Cord (12%), and Sturge Weber syndrome (17%) clinic were 3.86–7.37 time more likely to report difficulty in accessing surgeries compared to respondents from CDL (3%) (p < 0.001) and inability to obtain surgeries . A significant main effect for time (early vs. later pandemic) was only observed in the ability to access surgeries over time (p = 0.027); more respondents reported difficulty accessing surgeries between May and August (39.4%) than between September and December (13.6%).Twenty percent (CDL (3%) . Those fSix of 7 program directors responded to a survey assessing their experience utilizing the Pandemic Intake survey . Five ouWe successfully implemented a pre-visit Pandemic Intake questionnaire to quickly and systematically inform provider awareness of COVID-19 related patient concerns; the majority of program directors reported that this information helped them provide optimal care. Guardians of patients from more medically complicated clinics were significantly more likely to report perceived increased risk of contracting COVID-19, and the inability to access surgeries and therapies. Patients from CARD had higher reports of worsening behavior. Program directors worked to address these concerns and there was a significant decrease in the number of respondents unable to access surgeries in the later months of the pandemic (September to December). The implementation the Pandemic Intake survey facilitated the exchange of information and ideas, to benefit both patients and providers, and can be adapted to be used as a model for future pandemics at similar institutions.Perhaps due to their increased risk of co-morbidities , parentsThe pandemic's stay-at-home order significantly impacted mental health, routines and quality of life. Reports show higher rates of anxiety, depression, psychological distress and stress . LimitatThis study has important limitations. While the overall process of disseminating questionnaires was the same across programs, there were differences in the details of execution, like, in how many days before the clinic appointment the questionnaires were sent, which may have impacted participation. Families with infrequent access to email and non-English speaking families may have been less likely to participate. This was a cross-sectional study; further work is needed to understand implementation over time during the pandemic.This approach, and Pandemic Intake Questionnaire, may prove useful as the COVID-19 pandemic situation evolves, and for future pandemics, both at the Kennedy Krieger Institute and at other similar medical facilities.


The relationship between vitamin D and sarcopenia was inconsistent between men and women. Physical activity (PA) may interact with vitamin D on sarcopenia. However, the sex-specific relationships of vitamin D, PA and sarcopenia have yet elucidated. We aimed to examine the sex differences in the relation between vitamin D status, PA levels, obesity and sarcopenia in community-dwelling middle-aged and older adults, as well as whether vitamin D status is a modifier in the relationship between PA and sarcopenia.The current study was a cross-sectional study based on the baseline survey of the West China Health and Aging Trend (WCHAT) study. A total of 3713 participants aged ≥ 50y were included in our study. Sarcopenia was defined according to the Asian Working Group for Sarcopenia (AWGS) 2019 consensus. Obesity was defined by body mass index (BMI) (≥ 28 kg/m2) and body fat mass percentage (≥ 60th percentile in each sex group). 25-hydroxyvitamin D was measured by chemiluminescent microparticle immunoassay and PA was evaluated by a validated China Leisure Time Physical Activity Questionnaire (CLTPAQ). Multinomial logistic regression was performed to examine the relationship between PA, vitamin D and sarcopenia and obesity.p < 0.01). Vitamin D deficiency was only associated with sarcopenia in men . Low PA was significantly associated with obesity, sarcopenia, and sarcopenic obesity only in participants with serum 25(OH)D < 20 ng/ml.Low PA was significantly associated with higher odds of sarcopenia in women only (OR = 1.70,95%CI:1.18,2.46, The role of vitamin D and PA in obesity and sarcopenia was different between men and women, and the relationship between PA and sarcopenia was modified by serum vitamin D status. These findings highlighted the need to supplement vitamin D in individuals with physical inactivity and provide different interventions strategies to sarcopenia in men and women.Clinical trial number: ChiCTR1800018895.The online version contains supplementary material available at 10.1186/s12877-022-03577-4. Sarcopenia is an age-related skeletal muscle disorder defined as accelerated loss of muscle mass and function . SarcopePhysical inactivity and vitamin D deficiency often co-exist in older adults, and they are both risk factors for sarcopenia and obesity , 4, 5. PPhysical inactivity and vitamin D deficiency may have synergistic effects on muscle degradation and exacerbate sarcopenia . A previTherefore, the major purpose of this study was to examine whether the role of physical activity and vitamin D in sarcopenia, obesity, and sarcopenic obesity was different between men and women. The secondary objective was to investigate the relationship of physical activity and sarcopenia by different vitamin D status to examine whether vitamin D status is a modifier in this relationship.Data used in the present study are from the baseline survey of West China Health and Aging Trend (WCHAT) study . A totalAfter excluding 3474 participants who have missing data on grip strength, skeletal muscle mass index (SMI) and Short Physical Performance Battery (SPPB), 74 missing data on vitamin D, and 275 missing data on covariates, 3713 participants aged 50 years and older were included in the final analysis.We define obesity based on two different obesity markers: a) body mass index (BMI) ≥ 28.0 kg/m2 ; b) bodySarcopenia was defined according to the Asian Working Group for Sarcopenia (AWGS) 2019 consensus , which iParticipants were categorized into four groups: 1) Normal (N): neither obesity nor sarcopenia; 2) Obesity (O): only presence of obesity; 3) Sarcopenia (S): only presence of sarcopenia; 4) Sarcopenic obesity (SO): both presence of obesity and sarcopenia.Self-reported physical activity (PA) was evaluated by a validated China Leisure Time Physical Activity Questionnaire (CLTPAQ) , which wVenous blood samples were collected after an overnight fast to measure biochemical parameters. Serum 25(OH)D concentration (ng/mL) was performed using chemiluminescent microparticle immunoassay . Participants were classified into two groups according to serum vitamin D concentration: vitamin D deficiency (25(OH)D < 20 ng/ml) and vitamin D insufficiency or sufficiency (25(OH)D ≥ 20 ng/ml). All laboratory evaluations were performed by trained clinical laboratory technicians in accordance with standard operating procedures in hospital laboratories.Demographics and lifestyle data including age, sex, ethnicity, education , marital status , smoking history were collected. Self-reported diagnosed chronic diseases, including hypertension, diabetes mellitus, coronary heart diseases, chronic obstructive pulmonary diseases (COPD), arthritis, stroke, and tumor were also interviewed. We also included disability in activities of daily living (ADL disability). ADL disability was defined as having needs for assistance or difficulty in one or more of the ten items in Barthel Index . NutritiStatistical analysis was performed using Stata15.1 . We investigated participants’ characteristics using descriptive statistics and appropriate statistical tests. For descriptive analysis, continuous variables are expressed as the mean ± standard deviation (SD), and categorical variables are expressed as number (%). Group differences were evaluated using the Student’s t-test for continuous variables and the chi-squared tests for categorical variables.p < 0.05 was considered statistically significant.We used multinomial logistic regression to examine the relationship between PA levels , vitamin D status , and obesity or sarcopenia status . We also conducted sex subgroup analysis to explore the different role of PA and vitamin D in obesity and sarcopenia in men and women. Furthermore, we investigated the relationship of PA and obesity/sarcopenia at different vitamin D status to explore whether vitamin D could modify this relationship. All regression models were adjusted for age, sex, ethnicity, education, marital status, smoking history, common chronic diseases, ADL disability, and nutrition status. Two-sided A total of 3713 participants aged 50 years and older were included our analysis. The mean age was 61.9 years (SD 8.0), and 42% were aged < 60 years (Table p < 0.001). Vitamin D deficiency was significantly associated with obesity, sarcopenia, and sarcopenic obesity . In subgroup analysis, low PA was significantly associated with sarcopenia only in women. Vitamin D deficiency was independently associated with obesity, sarcopenia, and sarcopenic obesity in men, while significant association was only observed for obesity and sarcopenic obesity in women.Table p < 0.05, OR = 1.96, p < 0.001 and OR = 1.85, p < 0.05, respectively).Table The present study adds evidence to the sex-specific relationships between vitamin D status, PA levels, sarcopenia, obesity and sarcopenic obesity among middle aged and older people in west China. Vitamin D deficiency, defined as total 25(OH)D < 20 ng/ml, was found to be associated with sarcopenia only in men, and low PA was found to be associated with greater odds of sarcopenia only in women. Vitamin D deficiency was independently associated with obesity and sarcopenic obesity both in men and women. We also found vitamin D status modified the association between PA and sarcopenia. Specifically, among participants with 25(OH)D < 20 ng/ml, low PA was associated with increased risk of sarcopenia, whereas there was no significant association among those with 25(OH)D ≥ 20 ng/ml.Consistent with previous studies, our study confirmed that physical inactivity and vitamin D deficiency were associated with sarcopenia , 28. AddCompared with the role of vitamin D in sarcopenia development in men, physical inactivity was the main contributor to developing sarcopenia in women. Previous studies have found that low PA was significantly associated with sarcopenia , 33. In Other possible factors cannot be completely ruled out, and more studies are still needed to further prove.Low serum vitamin D was found to be associated with obesity and sarcopenic obesity regardless of sex. The inverse association between vitamin D and obesity has been established in the literature . A largeSerum vitamin D status was an important modifier in the relationship between PA and sarcopenia. As shown in our results, participants with low PA had higher odds of sarcopenia and sarcopenic obesity when participants had vitamin D deficiency. However, low PA was not significantly associated with sarcopenia and sarcopenic obesity among participants with higher concentration of vitamin D (25(OH)D ≥ 20 ng/ml). Another cross-sectional study conducted by Yang et al. found that vitamin D and PA had an interactive effect on sarcopenia. The effects of vitamin D on sarcopenia may be affected by the level of PA, and the effects of PA may also be affected by vitamin D levels . Yang etOur study had some strengths. First, WCHAT is a relatively large population-based study, and complete information on physical and health conditions was collected through standardized and structured interviews. Second, diagnosis of sarcopenia was based on the AWGS 2019 consensus, and definition of obesity was based on the combination of BMI and fat mass percentage assessed by BIA, which were more accurate and standard. However, several limitations should be considered in our current study when interpreting the results. First, cross-sectional analyses cannot elucidate any determination of the temporal nature of the associations between variables, for which longitudinal studies are required. Second, PA evaluation was based on self-reporting not accelerometer-measured physical activities, so potential recall bias may exist. Finally, our study population were from multiple ethnic groups. Although we adjusted for ethnicity in the regression models, the differences in vitamin D levels and PA habits between ethnic groups and whether these differences influence the relationships of them are not clear.This study has several implications for prevention and intervention to sarcopenia in older people. Firstly, it is recommended that simultaneous screening for vitamin D levels and evaluating levels of PA in older adults. Older adults should strive to avoid both physical inactivity and vitamin D deficiency. Secondly, supplementing vitamin D may help reduce the risk of sarcopenia in individuals with physical inactivity or physical limitations. Thirdly, women are encouraged to increase PA, while men should maintain adequate vitamin D levels. In the future clinical trials for sarcopenia, the intervention strategies for men and women should be different or individualized.The role of vitamin D and PA in sarcopenia was different between men and women, and the relationship between PA and sarcopenia modified by serum vitamin D status. These findings highlighted the need to supplement vitamin D in individuals with physical inactivity and provide different interventions strategies to sarcopenia in men and women.Additional file 1: Table S1. Associations between physical activity, vitamin D and obesity/sarcopenia according to age group. Table S2. Vitamin D levels of the total sample and according to physical activity level.


P < 0.001; Q3 vs. Q1, OR 0.8, 95% CI 0.6–1.0, P = 0.035; Q4 vs. Q1, OR 1.6, 95% CI 1.1–2.3, P = 0.008). The association remained stable in the fully adjusted model. A significant U-shaped association between corrected serum calcium and in-hospital mortality was observed in piecewise linear regression model . In conclusion, both decreased and increased corrected serum calcium is associated with increased in-hospital mortality in patients with AMI, and patients may have the lowest risk of in-hospital death when corrected serum calcium is 9.4 mg/dL (2.35 mmol/L).The association between serum calcium levels and the prognosis of patients with acute myocardial infarction (AMI) remains controversial. This study aimed to explore the association between serum calcium and in-hospital mortality in patients with AMI. The data of this study were extracted from the Philips eICU Collaborative Research Database. A total of 7284 patients were eventually enrolled in this study, of which 799 (10.97%) died during hospitalization. For each patient, serum calcium, corrected to albumin, was calculated and categorized into four groups: Q1 ≤ 8.5, Q2 8.5–9.5, Q3 9.5–10.5, and Q4 > 10.5 mg/dL. Multivariate analysis demonstrated that corrected sCa was an independent predictor of in-hospital death (Q2 vs. Q1, OR 0.5, 95% CI 0.4–0.7,  Calcium homeostasis is critical for maintaining normal muscle contractility, vascular tone, and electrical signaling1. An increasing number of people take calcium supplements as they are able to prevent osteoporosis and fractures3. Recently, a rising body of studies has suggested that serum calcium levels are associated with cardiovascular disease11. In addition, a recent study showed that the intermountain risk score, which includes calcium, is independently related to both short- and long-term mortality among AMI patients12. However, the clear relationship between serum calcium and mortality in patients with AMI is controversial. Most previous studies have found that low serum calcium is associated with higher mortality in patients with AMI, whereas high serum calcium is not associated with higher mortality13. However, some recent studies have found that high serum calcium is also associated with higher mortality in AMI patients, and we found that these studies included more patients with hypercalcemia14. Therefore, we speculate that both high and low serum calcium might be associated with higher mortality in AMI patients. This study further explored the association between serum calcium and in-hospital mortality in a large cohort of patients with AMI.Calcium is the most abundant mineral in the human body, 99% of which is stored in bones and teeth15. This database consists of vital sign measurements, care plans, the severity of illness measures, laboratory tests, diagnosis information, treatment information, and more. Before requesting access to the eICU Collaborative Research Database, you must complete the CITI “Data or Specimens Only Research” course. Then you can apply for registration to obtain permission to use the eICU Collaborative Database. After the registration application has been approved, the data will be disclosed. If the protocol is approved, the data can be directly downloaded from the eICU Collaborative Database (http://eicu-crd.mit.edu). The Massachusetts Institute of Technology  approved this data collection and waived informed consent requirements. This study's first author (Dingfeng Fang) has approved access to the eICU Collaborative Database . The authors are grateful to the original study group for providing data for the current analysis. The study report follows the broad EQUATOR guidelines16. All methods in our study were performed under the Declaration of Helsinki.The data of this study were extracted from the Philips eICU Collaborative Research Database. The database was a multi-center intensive care unit (ICU) database for over 200,000 admissions in 2014 and 2015 from 335 ICUs in 208 hospitals in the United States of AmericaThis study is a retrospective cohort study based on the eICU Collaborative Research Database . Patients with a diagnosis of AMI were included (N = 9884). All AMI patients were continuously enrolled unless: 1) serum calcium level was unrecorded; (2) albumin level was unrecorded. 7284 AMI patients were included in the final data analysis. The details of inclusions and exclusions were showed in Fig.  serum ca17. Demographic data, medical history, laboratory tests, and treatment information were extracted from the database.The primary clinical outcome of this study was all-cause in-hospital death. To make the data more reliable, serum calcium (mg/ml) was corrected to albumin (g/dL) using the Payne formula: corrected serum calcium (corrected sCa) = Serum calcium + 0.8 × Each patient's corrected sCa was calculated and categorized into four groups: ≤ 8.5, 8.5–9.5, 9.5–10.5, and > 10.5 mg/dL. Normally distributed continuous variables were presented as mean ± SD. Skewed distributed continuous variables were presented as median [interquartile range]. The categorical variables were presented as numbers (n) and percentages (%). The characteristics of this study population according to the corrected serum calcium levels were compared using a Kruskal–Wallis test or one-way analysis of variance (ANOVA) for continuous variables and a Chi-Square test for categorical variables. We performed univariate analysis to detect the possible risks associated with in-hospital death. Multiple logistic regression models were performed to evaluate the association between corrected calcium and in-hospital death. Multivariate adjusted models were applied as well as non-adjusted models. The adjusted confounders were selected basis on their association with the primary outcome. Different stratification was adjusted for different numbers of confounding variables. Furthermore, the threshold effect of corrected sCa on in-hospital death was explored using piecewise linear regression according to the smoothing plot.P value less than 0.05 was considered significant for all tests. Analysis was performed using the statistical software packages R  and Empower Stats .A As shown in Fig. P < 0.001; Q3 vs. Q1, OR 0.8, 95% CI 0.6–1.0, P = 0.035; Q4 vs. Q1, OR 1.6, 95% CI 1.1–2.3, P = 0.008). After adjusting for age, sex, BMI, and chronic renal insufficiency, the association between corrected sCa and in-hospital death was consistent with Model 1 . Furthermore, this association remained stable when age, sex, BMI, chronic renal insufficiency, COPD, diabetes mellitus, stroke, history of malignancy, hemoglobin, TC, LCL-C, HDL-C, lipid-lowering drugs, antiplatelet, anticoagulants, and nitroglycerin were adjusted in Model 3 .The details of univariate analysis in Table P < 0.001). The occurrence of in-hospital death increased with an increase in corrected sCa when the corrected sCa > 9.4 mg/dL .After the possible factors related to in-hospital death were adjusted, a U-shaped relationship between corrected serum calcium levels and in-hospital mortality in patients with AMI was observed Table ; Fig. 2.P < 0.001; corrected sCa > 9.4 mg/dL, OR 1.5, 95% CI 1.3–1.8, P < 0.001).This study retrospectively analyzed the association between corrected sCa levels and in-hospital mortality in patients with AMI. To analyze the in-hospital survival of 7284 patients with AMI, we found a U-shaped association between patients' corrected sCa levels on admission and in-hospital mortality. The results suggest that both high and low corrected sCa levels are associated with a higher risk of in-hospital mortality , which further elucidates the relationship between serum calcium and in-hospital mortality in patients with AMI. A meta-analysis of prospective cohort studies18 found a U-shaped relationship between dietary calcium intake and cardiovascular mortality , which may correspond to our study.Our findings are consistent with Shiyovich A et al.’s recent studies4. Consistent with Timo Schmitz et al., multiple previous studies have not found that high serum calcium is associated with an increased risk of in-hospital mortality in patients with AMI13. An 8-year, single-center study in China6 found that higher serum calcium was associated with a lower risk of in-hospital death . Thus, studies that are inconsistent with our study are not uncommon. We speculate that the main reasons for the different results are: (1) the study population is different; (2) the adjusted variables are different; (3) the statistical methods used are different. However, it is worth noting that our data include more hypercalcemia patients  because our study mainly focused on severe cases from the ICU of the United States of America. Therefore, our data may be more representative.The relationship between serum calcium and in-hospital mortality in patients with myocardial infarction remains controversial. In a 2021 study by Timo Schmitz et al., low serum calcium is associated with higher long-term mortality in patients with AMI, whereas high serum calcium was not13. In addition, patients with low serum calcium levels are more prone to ventricular arrhythmias and cardiac arrest20. (2) Low serum calcium is associated with multiple cardiovascular risk factors such as hypertension and left ventricular systolic dysfunction22. (3) Low calcium may lead to reversible heart failure and cardiomyopathy24. The mechanisms by which hypercalcemia leads to higher in-hospital mortality in patients with AMI may include the following: (1) High serum calcium levels may lead to subendothelial calcium deposition, leading to atherosclerosis25. (2) High serum calcium is associated with vascular calcification by binding to calcium-sensing receptors and inducing mineralization of smooth muscle cells26. (3) High serum calcium level increases the risk of heart failure, stroke, and type 2 diabetes mellitus28. One possibility deserves our attention: AMI leads to changes in serum calcium. However, this possibility is minimal because many prospective studies have found an association between serum calcium and in-hospital mortality in patients with AMI31.The mechanism responsible for the U-shaped relationship between serum calcium levels and in-hospital mortality in AMI patients is unclear. Several mechanisms may explain that low serum calcium is associated with higher in-hospital mortality in patients with AMI: (1) Serum calcium levels are directly related to the electrophysiological characteristic of the cardiomyocyte membrane. Low serum calcium may cause delayed closure of calcium channels, leading to prolonged plateaus35. However, it is worth noting that research in recent years has shown that calcium intake from the diet can benefit cardiovascular health. A geospatial analysis found an inverse association between high calcium spring water and cardiovascular mortality, citing waters with high calcium content as cardiovascular protective36. Multiple studies on drinking water have found the same result38. A meta-analysis found a U-shaped relationship between dietary calcium intake and cardiovascular mortality, and calcium intake ≥ 800 mg/d is associated with increased cardiovascular risk. Therefore, supplementing calcium in the diet may be beneficial, but only in moderation. In contrast, calcium intake from calcium supplements was seemly associated with an increased risk of cardiovascular death41. An IVUS study by Najdat Bazarbashi et al. found that oral calcium supplements may increase calcium deposition in coronary vessels, leading to poor prognosis42. The mechanism of oral calcium supplements increasing cardiovascular risk is unclear, likely associated with the sudden increase in serum calcium. However, it is worth noting that the symptoms of severe AMI patients with low serum calcium significantly improved after calcium supplementation5. Based on these controversies, our study has great clinical value for managing patients with AMI. For the first time, our study demonstrated the U-shaped association between corrected serum calcium and in-hospital mortality in AMI patients with a smooth curve and identified the inflection point of the curve (corrected sCa = 9.4 mg/dL). Clinicians can refer to our findings to formulate calcium supplementation strategies for AMI patients, thereby reducing patient mortality.Whether calcium supplementation can improve prognosis in patients with AMI remains controversialThis study has the following advantages: (1) The sample size is relatively large, and the serum calcium level of the patients was corrected using albumin, which makes the results more reliable; (2) The population of this study is critically ill patients from the ICU of the United States including more patients with abnormal serum calcium; therefore, our data are more representative; (3) This study is the first time to show the relationship between serum calcium and in-hospital death in AMI patients with a smooth curve and to determine the inflection point of the curve, which has an immense clinical value. However, this study excluded patients other than AMI and may not apply to all populations. In addition, due to the nature of observational studies, we can only observe associations, not causality. Our data come from critically ill patients in US ICUs with complex conditions. Although we have adjusted for confounders that may have been associated with the results, we cannot rule out some undocumented confounders confounding the results. Therefore, it is necessary to conduct higher-level clinical studies in more populations in the future to validate our findings.The corrected serum calcium level is an independent prognostic marker of in-hospital mortality in patients with AMI with a U-shaped association (inflection point: corrected sCa = 9.4 mg/dL). It is necessary to carry out clinical studies with a higher level of evidence to validate our findings in the future.


We aimed to investigate the aetiology and outcomes of illnesses in patients presenting to an emergency department after travelling to a malaria-endemic country, in order to raise awareness of both tropical and cosmopolitan diseases.A retrospective chart review was performed for all patients who underwent blood smear testing for malaria at the Emergency Department of the University Hospitals Leuven from 2017 to 2020. Patient characteristics, results of laboratory and radiological examinations, diagnoses, disease course and outcome were collected and analysed.A total of 253 patients were included in the study. The majority of ill travellers returned from Sub-Saharan Africa (68.4%) and Southeast Asia (19.4%). Their diagnoses fell into three major syndrome categories: systemic febrile illness (30.8%), inflammatory syndrome of unknown origin (23.3%) and acute diarrhoea (18.2%). Malaria (15.8%) was the most common specific diagnosis in patients with systemic febrile illness, followed by influenza (5.1%), rickettsiosis (3.2%), dengue (1.6%), enteric fever (0.8%), chikungunya (0.8%) and leptospirosis (0.8%). The presence of hyperbilirubinemia and thrombocytopenia increased the probability of malaria, with a likelihood ratio of 4.01 and 6.03, respectively. Seven patients (2.8%) were treated in the intensive care unit, and none died.Systemic febrile illness, inflammatory syndrome of unknown origin and acute diarrhoea were the three major syndromic categories in returning travellers presenting to our emergency department after a stay in a malaria-endemic country. Malaria was the most common specific diagnosis in patients with systemic febrile illness. None of the patients died.The online version contains supplementary material available at 10.1186/s40794-023-00190-0. Until early 2020, international travel was increasing steadily. In 2018, more than 1.4 billion international trips were undertaken , 4. MalaThe emergency physician faces several challenges: most importantly, life-threatening diseases, such as malaria, must be recognised and treated adequately in a timely manner. Yet there is a knowledge gap in the diagnosis and management of patients with tropical diseases, leading to increased mortality and morbidity . MoreoveThe aim of this study was to investigate the aetiology and outcomes of illnesses in patients presenting to the emergency department (ED) after a stay in a malaria-endemic country, in order to raise awareness of both tropical and cosmopolitan diseases.This retrospective observational study was conducted at the University Hospitals Leuven, Belgium, a tertiary care referral hospital. A computer-assisted search for this chart review was performed to identify all patients who underwent diagnostic blood smear testing for malaria during a three-year period, from 28/02/2017 to 07/03/2020. We chose blood smear testing as a surrogate for illness after a stay in a malaria-endemic, as this test was part of the diagnostic workup for ill travellers returning from these countries [The following information was retrieved from the medical records: demographic data ; travel history ; reason for travel ; prophylaxis and adherence to malaria treatment (non-adherence was defined as not following the prescribed medication regimen); referral source . Duration and onset of symptoms, intake of antimicrobial therapy before presentation, symptoms at presentation (fever (reported or measured in the ED), isolated fever , respiratory symptoms, ear, nose and throat abnormalities, anorexia, diarrhoea, dysuria or urinary frequency, myalgia and arthralgia) were recorded. Relevant physical findings  were collected from patient files. All available laboratory results  were reviewed. Results of all performed radiological examinations were registered. Also records of follow-up care  were reviewed for information on diagnosis, disease evolution and outcome.Travel destinations were grouped in eight global regions corresponding to those defined for the GeoSentinel Surveillance Network: Sub-Saharan Africa, the Middle East and North Africa, Southeast Asia, South-Central Asia, Northeast Asia, Latin America and the Caribbean, North America and Australia, New Zealand and Oceania . When diDiagnoses were made in accordance with internationally recognised case definitions . All diaCategorical variables are reported as totals and frequencies, and continuous variables are expressed as medians with interquartile ranges (IQR). We used 2 × 2 tables to calculate sensitivity, specificity, positive and negative likelihood ratios of signs, symptoms and laboratory findings. The confidence intervals (CI) for sensitivity, specificity and accuracy were 'exact' Clopper-Pearson confidence intervals.The CI of the likelihood ratios were calculated using the log method . Data wen = 39), repeated blood smears from the same patient (n = 38), absence of travel to malaria-endemic country within the past year (n = 26), incomplete patient records (n = 4), blood smear taken outside the ED (n = 1) or being asymptomatic (n = 1).A total of 362 blood smears from the returning travellers in our ED were performed in the predefined period, and 109 blood smears were excluded from further analysis because of failure to meet the age criterion . Detected bacteria included Salmonella Paratyphi A (n = 1), Salmonella Paratyphi B (n = 1), Salmonella group B (n = 1), Salmonella group D (n = 1), Escherichia coli (n = 3), Streptococcus agalactiae (n = 1) and Streptococcus pneumoniae (n = 1). The other three positive cultures were contaminated with coagulase negative staphylococci (n = 1), Acinetobacter Iwoffii (n = 1) and Corynebacterium spp. (n = 1). Urine cultures turned positive in 5.2% (10/192). Stool cultures were positive in 13 out of 38 tests: isolated pathogens consisted of Salmonella spp. (6/13), Shigella spp. (5/13) and Campylobacter spp. (2/13). All 54 patients that were tested for HIV were negative. Only 12 (6.9%) of the 175 chest X-rays performed were abnormal, showing an infiltrate (n = 10), pleural effusion (n = 1) or pulmonary nodule (n = 1). Abdominal ultrasound was performed in 138 patients (54.5%) and showed abnormalities in 23.9% of the examinations, including splenomegaly (n = 10), hepatosplenomegaly (n = 6), hepatomegaly (n = 3), lymphadenopathy (n = 3), colitis (n = 7), signs of enteritis (n = 3), acute appendicitis (n = 1) and sedimentation at the bladder base (n = 1). In 1 patient, 2 abnormalities were detected .Blood cultures were obtained from 231 out of 253 patients and were positive in 5.2% (n = 2), subarachnoid haemorrhage (n = 1) and lymphoproliferative disorder (n = 1).Table n = 33) or non-adherent (n = 7) with the prophylaxis (early discontinuation). In our study 5/40  had a negative blood smear. In four of these patients the rapid antigen detection test  turned positive (presence of histidine-rich protein II detected). One patient needed a PCR test ) for diagnosis. Plasmodium falciparum was found in 75% of the malaria cases. Four patients  with falciparum malaria met the WHO criteria for severe malaria . The majority of patients diagnosed with malaria returned from Sub-Saharan Africa (95.0%) and their most frequently reported symptoms were fever (97.5%), headache (75.0%), myalgia (55.0%), anorexia (37.5%), arthralgia (25.0%), abdominal pain (20.0%) and diarrhoea (17.5%). In three patients with a malaria diagnosis , a co-infection was detected (1 rickettsiosis (positive IgG antibodies to Rickettsia conorii), 1 acute hepatitis E and 1 influenza B).Malaria was detected in 40 patients (15.8%), all of them not taking malaria prophylaxis  and 6.03 (95% CI: 4.08 – 8.90), respectively (Table The majority of the 253 patients (55.7%) were treated in ambulatory care setting; 44.3% required admission to the hospital, and 2.8% were admitted to the intensive care unit. The median length of hospital stay was 4 days (IQR 3–6). None of the patients died.This study focusses on characteristics and outcomes of ill returning travellers presenting to the emergency department after a stay in a malaria-endemic country.n = 40) was the most common specific diagnosis in the patients with systemic febrile illness, followed by influenza (n = 13) and rickettsiosis (n = 8). Plasmodium falciparum, the cause of severe  malaria, was identified in 75.0% of the patients with malaria. Compared to the study of Siikamaki et al., who performed a similar retrospective study in the ED looking at febrile adults after travel to malaria-endemic areas, our data show a higher percentage of Plasmodium falciparum malaria (11.9% vs 3.5%) [Malaria (osis n = . Plasmodvs 3.5%) . Althougosis n = . Plasmodenza n =  and rickEarly diagnosis and treatment of malaria is of utmost importance, since delay in diagnosis is associated with fatal outcome . When anSalmonella Enteritidis and E. coli)) [It is important to stress that malaria may be acquired together with other pathogens that are endemic in the visited areas , and that symptoms can overlap. Therefore, additional diagnoses should always be considered, even when a diagnosis of malaria has already been made: a tropical co-infection may be present, may require specific therapy and may cause significant morbidity when left untreated . In our . coli)) .Although recognition and early treatment of life-threatening tropical diseases by the emergency physician is crucial, in our study only 22.5% of the patients had a ‘tropical’ diagnosis  was almost exclusively seen after return from Sub-Saharan Africa (7 of 8 patients), especially South-Africa (6/8), based on epidemiological data presumably 6 cases of African tick-bite fever. Enteric fever was only found in Asia in our study . Dengue was diagnosed in 4 patients (1.6%), all after travels to Asia or Latin America. These data are in accordance with the GeoSentinel surveillance data, and emphasize the importance of the region of return when determining the prior probability for a specific disease [When we looked at the prevalence of tropical disease according to geographical region of exposure, rickettsiosis  and/or positive IgG antibody titer to  disease .For a large proportion of the travellers presenting at the ED (23.3%), the aetiology of illness remained unclear, which is consistent with other reports in and outside the ED (no specific diagnosis in respectively 25.1 and 24.4% of patients) .Strongyloides hyperinfection syndromes [Mortality in returning travellers has been reported to range from 0.2 to 0.5% in other studies , 5, 7, ayndromes –21. In oSince this is a single-centred study performed in a tertiary care hospital, there are restrictions on generalisability to other non-tertiary centres. We used the blood smear test as a surrogate for illness in the returning travellers after a stay in a malaria-endemic country. Theoretically we may have missed some patients that erroneously did not have a blood smear, but we believe this is unlikely since training on imported diseases is organised on a yearly basis in our centre and the threshold to request a blood smear is low. We were not able to collect reliable data on pre-travel vaccination status and so we did not include it on our study. Furthermore inpatients presenting with diarrhoea; parasitic testing was not routinely performed. The indication was evaluated case by case. In our study, there were no diagnoses of parasitic diarrhoea (e.g. giardiasis or amebiasis) and this could be an underestimation. Finally, this study discusses the patient population before the onset of the Covid-19 pandemic. For now, it remains unclear if the pandemic will have a permanent influence on international travel and cause a shift in the epidemiology of diseases in returning travellers.Our study confirms the broad differential diagnoses of illnesses in returning travellers presenting at the ED after a stay in a malaria-endemic country, and thus the need for continued vigilance in the ED physician. Both tropical and non-tropical, infectious and non-infectious causes should be taken into account. Whether they are tropical or non-tropical, potentially life-threatening diseases should be excluded or confirmed first. A thorough clinical examination, directed laboratory tests and knowledge about the prevalence of tropical disease according to the geographical region of exposure can help narrow down the possibilities.Additional file 1. Final diagnoses.