The risks of death at 12 months and death or high dependency at 3 and 12 months in overweight  and obese patients  did not differ from normal-weight patients significantly after adjusting for baseline characteristics. Underweight patients had significantly increased risks of these three outcomes. In ischemic stroke patients, being overweight or obese was not associated with decreased mortality or better functional recovery but being underweight predicted unfavourable outcomes.The relation between obesity and stroke outcome has been disputed. This study was aimed to determine the association of body mass index (BMI) with mortality and functional outcome in patients with acute ischemic stroke. Data were from a national, multi-centre, prospective, hospital-based register: the ChinaQUEST  study. Of 4782 acute ischemic stroke patients, 282 were underweight (BMI < 18.5 kg/m While epidemiological investigations have concluded that obesity is strongly associated with an increased risk of stroke in the general population6, controversy exists regarding the relationship between obesity and outcome in patients who have had an established stroke18. Several articles report that increased body mass index (BMI) independently predicts a lower mortality after stroke or transient ischemic attack (TIA)15, but others have found no survival advantage in being overweight or obese after stroke18. Although Doehner et al. have found that obese patients have a better functional outcome than normal-weight patients following acute stroke15, the relation between BMI and functional disability is not well established. Such controversy raises doubts about the appropriateness of recommending weight loss for the secondary prevention of stroke in overweight and obese patients19. Moreover, most investigations have been restricted to Western countries16, raising further uncertainty about the importance of BMI in Asian stroke patients who have relatively low BMI but high stroke incidence than in other populations20.Obesity is an established risk factor for cardiovascular disease and poses a serious public health burden worldwideThe aim of our study was to investigate the association between BMI and stroke outcome, including mortality and functional measures, in a broad range of hospitalized patients with ischemic stroke in China.n = 62) study in 37 cities in China. The design and conduct of the ChinaQUEST has been described previously22. In brief, consecutive patients (aged ≥ 15 years) admitted to a hospital within 30 days from onset of acute stroke due to ischemic stroke or intracerebral hemorrhage were included during a 5-month period in 200622. Patients with subarachnoid hemorrhage were excluded.This study was on data from the ChinaQUEST , a multicentre, prospective, hospital-registry  and interviews with patients or their proxies (54%). Follow-up information about functional outcome as measured by the modified Rankin scale (mRS) was collected mainly by telephone interview (>80%) at 3 and 12 months. Death and cause of death were ascertained by family members (64.7%), a health professional (18.4%), records , death certificate (3.5%) or other sources (1.5%).Patients were assessed 4 times during the course of the study, at admission, discharge, and approximately 3 and 12 months post-strokeWritten informed consent was obtained from all patients or an appropriate family member (if the patient was unable to give it) to participate. The study was approved by the ethics committees of Peking University First Hospital (Beijing), Ruijin Hospital (Shanghai), Prince of Wales Hospital (Hong Kong), and The University of Sydney. Good Clinical Practice guidelines in accordance with the Declaration of Helsinki were used and the privacy of patients was strictly protected.24: underweight (BMI < 18.5 kg/m2), normal weight (BMI 18.5 to <24 kg/m2), overweight (BMI 24 to <28 kg/m2) and obese (BMI ≥ 28 kg/m2).BMI was calculated from height and weight (kilograms per square metre) measured by nurses or obtained from the patient or the accompanying relatives on admission. The patient population was sub-grouped into the following categories according to the Chinese Guidelines on Prevention and Control of Obesity in AdultsThe outcomes included all-cause mortality at 12 months, and a composite of death or high dependency at 3 and 12 months post-stroke. High dependency was defined as a mRS score of 3–5.Covariates considered included socio-demographic variables, medical history and clinical features of the index stroke, including: age, sex, partnership , education level  and annual household income ; medical history of hypertension, diabetes, hyperlipidaemia, atrial fibrillation, prior stroke/TIA, coronary artery disease  either self-reported or diagnosed in hospital post-stroke; prior dependency, current cigarette smoking, and regular alcohol consumption within the 3 months before stroke onset; time of presentation at the hospital , Glasgow coma scale (GCS) score on admission  and Oxfordshire Community Stroke Project (OCSP) classification .25, which defined underweight (BMI < 18.5 kg/m2), normal weight (BMI 18.5 to <25 kg/m2), overweight (BMI 25 to <30 kg/m2) and obese (BMI ≥ 30 kg/m2). Second, we restricted the study population to those who had not received thrombolysis treatment. Third, another definition of high dependency as a mRS score of 4–5 was used to validate the association between BMI and functional outcome15. Fourth, we performed a subgroup analysis according to smoking status (current smoker and current non-smoker). All P values were two-sided, with P < 0.05 considered statistically significant. All statistical analyses were performed using SPSS version 13.0 .Baseline characteristics were compared among different BMI subgroups using the one-way analysis of variance (ANOVA) and Chi-square test for continuous and for categorical variables as appropriate. The Cox proportional hazard model was performed to assess the association between BMI and all-cause mortality at 12 months. Similarly, logistic regression models were performed to evaluate the association between BMI and the composite outcome of death or high dependency at 3 and 12 months post-stroke. The association of BMI and high dependency was also evaluated in the survivors at 3 and 12 months after stroke. The distribution of causes of death in those who had died during the follow-up period was compared with Chi-square test among the BMI subgroups. We performed four additional analyses to assess the robustness of our main findings. First, we performed a sensitivity analysis using the World Health Organization (WHO) BMI classification criteria2. Of them, 282 patients (5.9%) were underweight, 2306 (48.2%) had normal weight, 1677 (35.1%) were overweight, and 517 (10.8%) were obese.There were 6508 acute stroke patients enrolled in the ChinaQUEST study. After excluding 1572 patients with intracerebral hemorrhage and 154 with uncertain diagnosis and missing data, a total of 4782 patients with acute ischemic stroke were included in our analysis. The mean age of those with ischemic stroke was 64.6 years (range 15–102 years) and 37.9% were female. Their average BMI was 23.8 ± 3.5 kg/mBaseline characteristics between the BMI subgroups are shown in Table There were 484 patients who had died within 12 months after stroke. High dependency was found in 1221 patients at 3 months and in 942 patients at 12 months. Unadjusted analysis showed obese patients did not differ from patients of normal BMI significantly in the risks for death and death or high dependency after stroke. Overweight patients had a slightly lower proportion of death or high dependency at 12 months than normal-weight patients. Those who were underweight had the highest risks of the three endpoints  nor obese  was associated with death at 12 months, and death or high dependency at 3 and 12 months significantly. Underweight patients remained at significantly highest risk of poor outcome  score was not collected in the ChinaQUEST database, we included GCS score and OCSP classification as confounders in our analyses, which had been used as measures of stroke severity in previous studies and had high inter-observer reliability35. Additionally, GCS score and OCSP classification had been validated as the independent predictors of stroke outcome in the ChinaQUEST cohort22.Stroke severity is closely associated with the prognosis after stroke3. To avoid the potential influence of smoking, we performed a subgroup analysis to assess the association of BMI and stroke outcome in current smokers and current non-smokers, respectively. The analysis showed there was no advantage of survival or functional recovery associated with overweight and obese in both subgroups, which supported our main findings.Some studies indicated smoking was an important confounder of the relation between BMI and mortality36. Further studies with measures of body fat distribution, such as waist circumference or waist-to-hip ratio, may provide a more accurate assessment of the association between obesity and stroke outcome.There are several limitations to the present study that should be addressed. First, our study is an observational investigation that suffers from the typical limitations of such studies. We adjusted the outcome analyses for a range of identified confounding variables; however, the findings may be influenced by unknown confounders. Second, we could not exclude the possibility of bias due to other treatment and intervention that might influence stroke outcome, although the sensitivity analysis showed that the intravenous thrombolysis treatment did not affect the results of our main analysis. Third, the definition of ‘overweight’ and ‘obesity’ was based on BMI in our study. However, BMI does not reflect body fat distribution, which is also important to the evaluation of obesityIn conclusion, the present study shows that there is no survival advantage of overweight or obesity after ischemic stroke. The risk of death and functional disability is not significantly different in overweight and obese patients as compared with normal-weight patients. Underweight is associated with the highest risk of death and high dependency after stroke. Our results support the rationality of weight control for overweight and obese patients recommended by current guidelines on secondary prevention of stroke. However, the observational nature of our study warrants further validation.


Serum exosomal proteins have great potential as indicators of disease status in cancer, inflammatory or metabolic diseases. The association of a fraction of various serum proteins such as carcinoembryonic antigen (CEA) with circulating exosomes has been debated. The establishment of a method to measure the exosomal fraction of such proteins might help resolve this controversy. The use of enzyme-linked immunosorbent assays (ELISAs) to measure serum exosomal molecules, for example CEA, is rare in research laboratories and totally absent in clinical biology. In this study, we optimized a method for assessment of serum exosomal molecules combining a treatment by volume-excluding polymers to isolate the exosomes, their subsequent solubilization in an assay buffer and ELISA.One hundred sixteen consecutive patients with colorectal cancer were enrolled for this study between June 2015 and June 2016 at Wakayama Medical University Hospital (WMUH). Whole blood samples were collected from patients during surgery. Exosomes were isolated using the ExoQuick reagent, solubilized in an assay buffer and subjected to CEA detection by ELISA. The procedure of serum exosome isolation and the formulation of the assay buffer used for the ELISA were optimized in order to improve the sensitivity and specificity of the assay.A five-fold increase in the concentration of the exosomes in the assay buffer  and the addition of bovine serum albumin (BSA) resulted in more accurate measurements of the serum exosomal CEA. The thawing temperature of frozen serum samples before exosome extraction was also optimized. A validation study that included one hundred sixteen patients with colorectal cancer demonstrated that serum exosomal CEA from samples thawed at 25°C exhibited a better AUC value, sensitivity, and specificity as well as a more correct classification than serum CEA.We optimized an easy and rapid detection method for assessment of serum exosomal CEA. The thawing temperature of frozen serum prior to exosome extraction, the formulation of the assay buffer used for exosome solubilization and the concentration of the exosomes in this buffer were fine-tuned to enable the appropriate and accurate measurement of serum exosomal CEA. An exosome is a small vesicle, measuring 40–150 nm in size . VariousExosomal miR-19a  and exosIn the present study, we optimized a method to measure serum exosomal CEA by utilizing volume-excluding polymers and ELISA, and we examined whether serum exosomal CEA can better predict the existence of distant metastasis than serum CEA.We enrolled 116 consecutive patients treated for colorectal cancer between June 2015 and June 2016 at Wakayama Medical University Hospital and 8 normal healthy subjects. Written informed consent was obtained from all subjects. The mean age of the patients was 68.9 years (range 42–92), and there were 66 males and 50 females. Based on the TNM classification system, 39, 22, 36, and 19 patients had colorectal carcinoma of stages I, II, III, and IV, respectively. All patients underwent surgery, during which whole blood samples were collected. The current study was approved by the Human Ethics Review Committee of Wakayama Medical University Hospital. All experiments were performed in accordance with relevant guidelines and regulations.g for 10 min at 4°C. Serum was distributed in 250-μl aliquots into Eppendorf tubes and frozen at –20°C until analysis. Frozen serum samples were thawed at 4, 25, or 37°C for 30 min. A total of 63 μl of ExoQuick Exosome Precipitation Solution  was added to each thawed 250-μl serum sample, which was then incubated at room temperature for 30 min. The serum/ExoQuick mixture was centrifuged at 1500 ×g for 30 min at 4°C. The supernatant was aspirated, and the exosome pellet was resuspended using water or 1% bovine serum albumin (BSA). A CEA (human) ELISA kit  was used in our assay.Whole blood was collected into serum-separating tubes and then centrifuged at 1000 ×To evaluate whether thawing temperature at 4, 25, or 37°C modulates the yield and purity of exosome, western blotting for CD63 as exosomal marker was performed. Frozen serum samples from normal healthy subject were thawed at 4, 25, or 37°C for 30 minutes. After exosome isolation by using ExoQuick Exosome Precipitation Solution, protein extraction was performed with RIPA lysis buffer . The samples were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis. In each lane, 10 μl of each sample was loaded and separated in a precast polyacrylamide gel . Proteins were then electrotransferred onto PVDF membrane . After blocking the membrane with 5% electrophoresis-grade nonfat milk, primary and secondary antibodies were incubated for 60 min each in a 5% milk solution. Immune complexes were visualized by incubating the membranes with an HRP-conjugated anti-mouse antibody using the ECL detection reagent . The primary antibody was mouse monoclonal CD63 antibody . The secondary antibody was sheep anti-mouse IgG conjugated to HRP .EXOCET Exosome Quantitation Kit(System Biosciences) and a CEA (human) ELISA kit (Abnova) were used for the quantitation of each exosome sample and the measurement of exosomal CEA concentrations, respectively.To optimize measurement of serum exosomal CEA, the effects of concentration of the samples, formulation of the solvent, and thawing temperatures for frozen samples were evaluated. Samples from eight patients and eight normal healthy subjects were used. Each patient’s exosome pellet was resuspended using 250 μl of Milli-Q water (1×), 50 μl of Milli-Q water (5×), 250 μl of 1% BSA (1×) or 50 μl of 1% BSA (5×). Frozen serum samples from the same participant were thawed at 4, 25, and 37°C. An Because serum CEA values are used to diagnose suspected colorectal cancer recurrence, serum exosomal CEA concentrations at each temperature were evaluated as a potential diagnostic marker indicating the existence of distant metastasis. Forty-eight patients were enrolled in this part of the study. Frozen serum samples from the same patient were thawed at 4, 25, and 37°C. Accuracy, sensitivity, specificity, and correct classification were evaluated.A validation study was conducted using samples from all 116 colorectal cancer patients. Frozen serum samples were thawed at an appropriate temperature obtained from the results based on the comparison described in the previous section. Accuracy, sensitivity, specificity, and correct classification were compared for serum exosomal CEA and serum CEA.P-value < 0.05 was considered to be statistically significant.The accuracy of serum exosomal CEA was measured using the area under the ROC curve. Sensitivity, specificity, and correct classification were also calculated from the curve. Statistical calculations were performed using the STATA software program, version 13 . Exosomal proteins such as CEA are present in small amounts, so high protein concentrations may be needed for their detection using ELISA. The present study demonstrated that ELISA could not detect a 1× CEA sample dissolved in Milli-Q water. Even when the exosome concentration was increased by a factor of five, serum exosomal CEA remained undetectable . This inThe efficacy of volume-excluding polymers depends on the hydrophilicity or hydrophobicity of the substrate. We hypothesized that the thawing temperature of frozen serum alters the hydrophilicity or hydrophobicity of exosomes and CEA molecules. Based on this hypothesis, frozen serum was thawed before exosome isolation with ExoQuick reagent. The yield in western blots and quantity of exosomes did not differ among the various thawing temperatures  ELISA kit. Neither the reagent nor the antibodies were prepared for the detection of exosomal markers. Exosome isolation reagents should be developed for specific exosomal markers. The measurement range for the CEA (human) ELISA kit used in this study was 0 to 120 ng/ml, which is unsuitable for detecting exosomal CEA, because the cut-off value for exosomal CEA was approximately 2 ng/ml in the current study. Appropriate exosome isolation reagents and ELISA kits specific for exosomal markers need to be improved and established for appropriate applications involving exosomal proteins and glycoproteins.In conclusion, we optimized a simple and rapid detection method for exosomal glycoproteins such as CEA. The concentration of the exosome sample, the formulation of the solvent for the exosomes, and the thawing temperature of frozen serum before extraction were fine-tuned to enable the appropriate and accurate measurement of exosomal CEA.


Since 2005, the Government of Ghana and its partners, in concerted efforts to control malaria, scaled up the use of artemisinin-based combination therapy (ACT) and insecticide-treated nets (ITNs). Beginning in 2011, a mass campaign of long-lasting insecticidal nets (LLINs) was implemented, targeting all the population. The impact of these interventions on malaria cases, admissions and deaths was assessed using data from district hospitals.Records of malaria cases and deaths and availability of ACT in 88 hospitals, as well as at district level, ITN distribution, and indoor residual spraying were reviewed. Annual proportion of the population potentially protected by ITNs was estimated with the assumption that each LLIN covered 1.8 persons for 3 years. Changes in trends of cases and deaths in 2015 were estimated by segmented log-linear regression, comparing trends in post-scale-up (2011–2015) with that of pre-scale-up (2005–2010) period. Trends of mortality in children under 5 years old from population-based household surveys were also compared with the trends observed in hospitals for the same time period.Among all ages, the number of outpatient malaria cases (confirmed and presumed) declined by 57%  by first half of 2015  compared to the pre-scale-up (2005–2010) period. The number of microscopically confirmed cases decreased by 53% (28–69%) while microscopic testing was stable. Test positivity rate (TPR) decreased by 41% (19–57%). The change in malaria admissions was insignificant while malaria deaths fell significantly by 65% (52–75%). In children under 5 years old, total malaria outpatient cases, admissions and deaths decreased by 50% (32–63%), 46% (19–75%) and 70% (49–82%), respectively. The proportion of outpatient malaria cases, admissions and deaths of all-cause conditions in both all ages and children under five also fell significantly by >30%. Similar decreases in the main malaria indicators were observed in the three epidemiological strata . All-cause admissions increased significantly in patients covered by the National Health Insurance Scheme (NHIS) compared to the non-insured. The non-malaria cases and non-malaria deaths increased or remained unchanged during the same period. All-cause mortality for children under 5 years old in household surveys, similar to those observed in the hospitals, declined by 43% between 2008 and 2014.The data provide compelling evidence of impact following LLIN mass campaigns targeting all ages since 2011, while maintaining other anti-malarial interventions. Malaria cases and deaths decreased by over 50 and 65%, respectively. The declines were stronger in children under five. Test positivity rate in all ages decreased by >40%. The decrease in malaria deaths was against a backdrop of increased admissions owing to free access to hospitalization through the NHIS. The study demonstrated that retrospective health facility-based data minimize reporting biases to assess effect of interventions. Malaria control in Ghana is dependent on sustained coverage of effective interventions and strengthened surveillance is vital to monitor progress of these investments. Ghana has an area of 238,537 sq km and a population of 27 million . MalariaSince 2000, the Government of Ghana accelerated malaria control efforts guided by three consecutive national malaria strategic plans covering: first, for the period 2000–2010; second, for 2008–2015; and, an updated one for 2014–2020. Funding for malaria control increased from <US$10 million in 2005 to >US$80 million in 2015 with the support of the Global Fund to Fight AIDS, Tuberculosis and Malaria (GFATM), the President Malaria Initiative (PMI), Department for International Development (DFID), and other development partners. The National Malaria Control Programme (NCMP), scaled up distribution of insecticide-treated nets (ITNs) and artemisinin-based combination therapy (ACT) as key interventions, and IRS in selected districts. For monitoring progress, Ghana’s disease surveillance relies on the District Health Management Information system (DHIMS-2). Data are aggregated by health facility and age with online access to authorized users. The DHIMS-2 is integrated for all diseases with an average reporting completeness of 95% by 2015 from the public and faith-based health centres and hospitals and only 62% from the private sector [In view of significant investments, the NMCP of Ghana and World Health Organization (WHO) assessed the impact of the interventions on trends of malaria cases and deaths using retrospective data collected from sample hospitals, representing the three epidemiological zones covering the period 2005–2015.Routine distribution of ITNs targeting children under 5 years old and pregnant women started in 2003 in 20 pilot districts and was scaled up nationwide during 2004–2010. During 2006–2009, >4.5 million ITNs were distributed through antenatal care (ANC), Child Health weeks, voucher schemes, and commercial sources. However, the strategy was replaced with a long-lasting insecticidal net (LLIN) mass campaign, starting at the end of 2010 with the goal of achieving universal coverage in all ages at a ratio of one net per two persons. During 2010–2012, >12 million LLINs were delivered, and another 12.3 million LLINs during 2013–2015. In 2012, the AngloGold Ashanti Malaria Control Programme  implemented indoor residual spraying (IRS) in seven districts, expanding to 22 districts in five regions  in 2015. PMI supported five districts for IRS in the Northern region. In total, in 2015, 4.7% of the entire population in the country benefited from IRS. This population also received LLINs.Parasitological testing of patients of age 5 years and above was primarily limited to hospitals. Since 2010, confirmation of all ages with either microscopy or rapid diagnostic tests (RDTs) has been introduced. Health centres and health posts use RDTs as primary diagnostic tool while higher facilities use microscopy, reserving RDTs for emergency cases, and for situations where microscopy is not functional or caseloads exceed microscopy diagnostic capacity. Therefore, only records of microscopic testing from the hospitals were used for this study.The country changed its first-line anti-malarial treatment from chloroquine to artesunate–amodiaquine (AS–AQ) in 2004 and added artemether–lumefantrine (AL) and dihydro-artemisinin–piperaquine (DHAP) as alternatives in 2009. Quinine was the principal medicine for severe malaria until 2010, when injectable artesunate was adopted as the drug of choice. The Affordable Medicines facility for Malaria (AMFm), launched in 2011, increased access to ACT in the private sector from 31% in 2010 to 83% in 2011 [For community-based intervention, between 2005 and 2012, nearly 2218 CHPS have been established in the country. The health staff in the CHIPS diagnose with RDTs and treat malaria and assist delivery at the community. However, presumptive treatment of malaria may occur for logistic reasons, when RDTs stock-out.Prior to 2007, patients of all ages were charged for malaria diagnosis and treatment, which was a major barrier to accessing basic health services for majority of the population. To address the problem of access, the NHIS was initiated in 2003 and became operational in 2005 to subsidize maternal and child health services, which became free in 2007. In the NHIS, urban and rural populations pay a premium of about US$17 and US$11 per year, respectively, and when the cost is beyond what NHIS cover, patients cover the difference. For people whose inability to pay is certified by local authorities, the Government covers all. Despite these provisions, the coverage of the NHIS remains at 38% owing to financial challenges to sustain the scheme, identification of the poor and vulnerable, identification (ID) card management, quality of care, and slow information system .Long-lasting insecticidal nets and IRS data were obtained from district records. The proportion of the population potentially protected by LLINs was calculated for each year, assuming each LLIN covered 1.8 persons and lasted 3 years. Prospective district population was derived from the 2010 Ghanaian census, using the United Nations growth rate for Ghana , 6. To mA WHO standardized protocol (unpublished) was used for hospital data collection. A total of 88 hospitals in three epidemiological zones were randomly sampled . Although the design was to select 30 representative hospitals from each zone, the savannah had only 28 hospitals and all were sampled. Data abstraction teams consisting of two persons visited each hospital for 2 days.Monthly summary reports were used as the main source of records for: (i) outpatient all-cause consultations and malaria cases; (ii) inpatient all-cause and malaria admissions, all-cause deaths, malaria deaths, anaemia admissions, and anaemia deaths; and, (iii) laboratory records for microscopically tested and positive cases. Where monthly summary records were missing, register books were used as primary source. Data were collected for two age groups: all ages and under 5 years of age.Analysis of microscopic tests and confirmed cases was possible only for all ages as records of microscopic results could not be disaggregated by age, outpatient or inpatients. Microscopically confirmed malaria cases, malaria admissions and malaria deaths were the main interest of the analysis. A confirmed malaria case for this study was defined as a blood slide positive for malaria. Malaria admission was defined as a microscopically confirmed inpatient case, diagnosed at discharge as severe malaria. However, some malaria admissions were admitted based on clinical signs suggestive of severe malaria. A malaria death was defined as death attributed to malaria among the admitted cases. All anaemia admissions were assumed to be severe anaemia (<7 mg/dl). The test positivity rate (TPR) was computed by dividing number of positive slides by the total number of slides examined.To control for other factors that might influence the observed trends, the following additional indicators were computed: (i) non-malaria outpatient consultations ; (ii) non-malaria admissions ; and, (iii) non-malaria deaths . In addition, data on admitted patients with or without health insurance were extracted from the DHMIS-2 to assess the difference in disease trends among those insured and non-insured. These allowed the assessment of whether changes in malaria cases and deaths were truly attributable to interventions or to changes in healthcare-seeking behaviours or other factors that affect all diseases in a similar way. In addition, the trends of child mortality from population-based surveys for Ghana were also reviewed and triangulated with the trends observed in the hospitals.To control for the potential effect of climatic changes in trends of malaria during pre- or post-scale-up periods, data on monthly rainfall in mm, minimum and maximum temperature in  °C by region, were obtained from the Ghanaian Meteorology Department.For filling missing monthly data, either of the following approaches were applied: (i) if the missing data were in the 1st year, then average the consecutive 2 years of the same month, (ii) if the missing data were in the last year, then average the two preceding years of the same month; and, (iii) if the missing data were in between 2 years, running averages of the preceding and consecutive year for the same month. Because the data points end in June 2015, monthly data were aggregated biannually (two periods for each year) making a total of 21 periods covering January 2005–June 2015.P < 0.05). This model corrects for autocorrelation [Assuming all other interventions constant, the effect of an LLIN mass campaign since 2011 was evaluated by comparing the disease trends of the pre-scale up years 2005–2010), i.e., period 1–11 with that of period 12–21 . Changes on trends were estimated as relative per cent change by comparing the observed value and predicted value at period 21 assuming a continuation of the pre-scale up time trend throughout period 21 if there were no interventions . The interpretation of the predicted relative change in percentage at period 21 therefore takes into account the trends (slop) of all the values during 2011 and 2015. This was done using a segmented regression model of an interrupted time series with breakpoint of period 11 , an appr, i.e., prelation  and adjuEighty-eight district hospitals had complete on outpatient and inpatient data and 70 on labortory data; and all were included in the analysis, each representing a district. Nearly 21,908 missing monthly values, representing 10% of the total 210,709 records for the key indicators, were imputed using either of the approaches described in the methods.® Olyset®, Interceptor, and Netprotect. The percentage of the hospitals with available stock of all forms of ACT was 100% during 2006–2012, and >95% during 2013-2015 . The number of microscopically confirmed cases had little change in observed values but a significant predicted decrease of 53% (28–69%), while the number of microscopic tests had an observed increase and little change in predicted values. The TPR in all ages declined by 30% (observed) and a predicted decrease of 41% (52–75%) nationwide during the same period. Malaria admissions showed 52% increase in observed trends and insignificant predicted change, 39% (0–63%). Malaria deaths fell by 49% in observed and predicted trends significantly by 65% (52–75%)  to 581,906 in the first half of 2015 (period 21), with an observed decrease of only 8%, but significant predicted decrease of 57%  during 2011–2015 compared to the trend of 2005In children under 5 years old, total malaria outpatient cases (both confirmed and presumed cases) had little change in observed values but a significant predicted decrease of 50% (32–63%). Malaria deaths had an observed 59% decrease and a significant predicted decline of 70% (49–82%). The number malaria admissions had an observed increase of 48% and insignificant predicted change of 46% 19–75%); the proportion of outpatient malaria cases, admissions and deaths of all-cause conditions showed an observed decline of 35, 39 and 56% respectively; and a significant predicted decline of 30% (21–37), 49% (40–57%) and 59% (40–72%)  decreased significantly by 16% (16–40%), 66% (30–83%), and 55% (9–77%), respectively. TPR decreased significantly by 33% (7–58%) in coastal and by 37% 2–61%) in savannah while the change in forest was insignificant. Malaria admissions in all ages showed little or insignificant change in forest and savannah but decreased significantly by 40% in coastal (14–58%). Malaria deaths declined significantly by >64% in all the three zones. For children under five, malaria admissions either remained unchanged or increased while malaria deaths fell by >57% across all three strata. Observed trends of inpatient anaemia cases in Coastal, Forest and Savannah increased by 32, 216 and 136% respectively while the anaemia-related deaths increased by only 37, 16 and 106%, respectively. All predicted trends of anaemia cases and deaths were statistically insignificant , the TPR in all ages decreased significantly by 89% (77–95%); malaria admissions and deaths decreased significantly by 68% (21–87%) and 88% (71–95%), respectively. On the other hand, the decrease in trends of malaria indicators in the non-IRS districts (34 within the same regions) was smaller. The TPR decreased only by 38% (16–54%); malaria admissions showed little change, 35% (−15 to 63%), and malaria deaths decreased by 44% (16–62%). The decreases in proportion of malaria outpatients, inpatients and deaths of all-cause conditions were much higher in the IRS districts compared to the non-IRS districts (Table P < 0.05)  Table .Fig. 6RaBetween 2002 and 2009, mixed LLIN distribution models, including free distribution to targeted groups, subsidized LLINs through the public, private sector and NGOs, and full-cost sales, resulted in moderate increase of ITN ownership in 2008 . The masFollowing the LLIN campaigns in 2010, this study in 88 hospital data showed a 53% decline in trends of microscopically confirmed malaria cases against a backdrop of increased or constant testing, 65% decrease in malaria deaths and 41% decrease in TPR in all ages. The number of outpatient malaria cases, and malaria deaths in children under 5 years, decreased by 50 and 70%, respectively. Malaria admissions increased during the study period. In all three epidemiological zones, outpatient malaria cases decreased by 50%, malaria deaths by 60%, and TPR by >30%. Malaria admissions in children under five increased during 2011–2015 across all epidemiological zones. The decrease in the main malaria indicators was higher in the regions where IRS was employed, i.e., malaria admissions (68%), malaria deaths (88%) and TPR (89%). Whereas, in non-IRS regions, malaria admissions increased by 35%, malaria deaths and TPR decreased by only 44 and 38%, respectively. All the key malaria indicators decreased significantly across all age groups and regions with different levels of baseline malaria burden in the face of increased healthcare utilization. The paradox with the increase in malaria admissions (particularly in children under five) during the post-scale-up period (2011–2015) can be explained by the increased service availability or access to free admission owing to the provision of NHIS. The twofold increase (126%) in the number of inpatient anaemia cases shows the strong association with malaria admissions accessing free inpatient services. The increase in inpatient anaemia was particularly prominent in the Forest and Savannah zones  observed in the health facilities , to 82 per 1000 live births during 2009–2011 (MICS 2011), and 60 per 1000 live births representing at least a decline of 43% between 2008 and 2014. The decrease in malaria deaths may have contributed to the decline in all-cause deaths in children under five, consistent to previous observations . HoweverCaution has to be made when interpreting the results of such health facility-based data often affected by changes in service utilization and reporting completeness . The anaFive years (since 2011) after universal distribution of LLINs targeted all ages while maintaining high coverage of all other interventions during both pre- and post-scale up periods, the study showed substantial decrease across all malaria indicators including: confirmed cases, test positivity rate and malaria death. Declines in malaria deaths were higher in children under five than in older age groups. The trends in malaria indicators seen at the health facilities evidently reflected the steep downward trend of all-cause child mortality at the population level. The climatic effect on the time trend of malaria indicators in Ghana seems to be significantly related to fluctuation in minimum temperature, the importance of which is reported elsewhere , 37. Inc


However, intrinsic chemical compositions of NPs caused inconsistent biodistribution and toxic profiles which attracted little attention. In this study, silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs) were used to determine the biodistribution, toxickinetic, and genotoxicity variances in murine animals. The results demonstrated AgNPs and AuNPs were primarily deposited in the mononuclear phagocyte system (MPS) such as the liver and spleen. In particular, AuNPs seemed to be prominently stored in the liver, whereas AgNPs preferentially accumulated in more organs such as the heart, lung, kidney, etc. Also, the circulation in the blood and fecal excretions showed higher AgNPs contents in comparison with the AuNPs. Measurements of the mouse body and organ mass, hematology and biochemistry evaluation, and histopathological examinations indicated slight toxic difference between the AgNPs and AuNPs over a period of two months. RT-qPCR data revealed that AgNPs induced greater changes in gene expression with relevance to oxidative stress, apoptosis, and ion transport. Our observations proved that the NPs chemical composition played a critical role in their in vivo biodistribution and toxicity.Nanoparticles (NPs) size, surface functionalization, and concentration were claimed to contribute to distribution and toxicity outcomes of NPs  Currently, silver nanoparticles (AgNPs) are one of the most widely used NPs in commercial products  because of their strong antimicrobial and anti-inflammatory properties3. Gold nanoparticles (AuNPs) offer a wide range of applications including cosmetics, chemical sensing, drug carriers, bioimaging, and gene therapy5. The extended use of these inorganic NPs can lead to frequent human exposure via various routes  during manufacture, use and disposal. Moreover, compared with their bulk counterparts, AgNPs and AuNPs hold greater surface area to volume ratio, higher particle reactivity, and can undergo surface modifications which could result in greater side effects once these are absorbed into the body6. It is therefore imperative to understand the potential biological responses to AgNPs and AuNPs in vivo.Inorganic nanoparticles (NPs) have attracted increasing attention and are used in many fields, such as biomedicine, various industries, and electronics due to their excellent physicochemical propertiesin vitro studies demonstrated that inorganic NPs can induce the production of reactive oxygen species (ROS), DNA damage, and apoptosis10. These observations resulted in a number of in vivo rodent studies that were focused on the toxicity or biokinetics of inorganic NPs following various administration routes33. Table 12. Major organs of accumulation for AgNPs have been shown to be the lungs and liver in a 90-day inhalation toxicity study23, and liver in several long term oral exposure studies24. Also, intratracheal injection showed that the majority of AuNPs were found in the lungs regardless of PEG surface modification25. Of note, during oral and inhalation exposures, or dermal contact, a few NPs were able to pass through the gastrointestinal tract (GIT), lung barriers, and dermis into the blood, resulted in extremely low amount of NPs translocated to the secondary organs and thus difficult to detect and compare the accumulation profile of the NPs in the whole body28. To avoid limited systemic exposure due to the cellular barriers present in the GIT, lung, and skin, the present study was used to repeated intravenous administration of two inorganic NPs to compare their toxicity and distribution profiles in mice.Indeed, previous in vivo studies in rodents through intravenous injection, which examined the distribution and/or toxicity of various types of AgNPs and AuNPs with sizes ranging from 1.4 nm to 250 nm were enumerated in Table 19. Meanwhile, they also exhibited that the major accumulation organs were observed in the liver and spleen, and the smaller NPs had a more widespread organ distribution27. More specifically, 10 nm AuNPs were found in various organs including the liver, spleen, kidney, heart, lungs, testis, brain, and thymus, whereas 250 nm AuNPs were restricted to the liver and spleen26. These studies have reported strong influences of exposure routes, animal gender, size, shape, concentration, surface coating, aggregation, stability, or purities of nanomaterials on the biodistribution of AgNPs and AuNPs22.The classical 29. Also, it was demonstrated that the silver concentration in organs was closely related with the Ag+ concentration in the AgNPs suspension which could be used to deduce that only silver ions passed through the GIT causing accumulation in the liver and spleen3. Both studies exhibited that the released silver ions and not the “nano-size” affected the NPs distribution and toxicity after oral exposure. Additionally, AgNPs and AuNPs were used to study whether the toxicity induced by NPs size or NPs chemical composition in a zebrafish model34, because both NPs possess similar chemical characteristics . Nevertheless, no rodent toxicity studies have shown possible biodistribution and/or toxicity profiles among NPs with different chemical compositions and identical surface coating such as AgNPs and AuNPs after intravenous administration, even though the NPs chemical composition, as well as particle size, exposure route, and surface chemistry, is an important parameter that contributed to the toxicological responses.However, it is still unclear whether distribution, translocation, and elimination of AgNPs and AuNPs would mainly result from the NPs size effects or NPs chemical composition (such as released the free ions). In a 28-day oral exposure study, silver acetate (Ag ions) showed higher organ distribution throughout the rat body compared with AgNPs19. Both carboxylic acid coated AgNPs and AuNPs in this study can be conjugated to peptide, protein, and other amine containing molecules for various biomedicine applications. Herein, as a function of chemical composition, the effects of AgNPs and AuNPs on toxicokinetic and toxicity were evaluated to understand their nanotoxicity.The aim of the present study was to determine the differential accumulation, distribution, toxicity, and potential genotoxicity of AgNPs and AuNPs in mice following repeated intravenous injection for 4 weeks, at a dose with limited systemic toxicity as reported in a relevant study35.As shown in Fig. 37. No significant changes in food consumption and body weight were observed in either of the animal groups treated with AgNPs or AuNPs. As shown in Fig. The food consumption, body weight, and organ index of each animal during the entire experimental period were evaluated as previously reportedAs shown in Fig. The kinetics of AgNPs and/or AuNPs in the blood and feces was determined by measuring the AgNPs or AuNPs concentrations in successively collected samples. Figure 38. As a result, we determined the standard inflammatory biochemical parameters such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), albumin (ALB), the ratio of albumin to globulin (A/G), gamma glutamyl transaminase (GGT), alkaline phosphatase (ALP), blood urea nitrogen (BUN), and urea (UA). These biochemical markers, except the GGT, ALP, and UA, in either AgNPs or AuNPs treated groups were not statistically different from the control groups as shown in Fig. in vivo and presented in Fig. 39 and ZnS and ZnO QDs40 and CdSe/ZnS QDs41; however, they were within the normal range that had been reported in the literature. Other biochemical and hematology data were included in the Supplementary information , and Zrt- and Irt-related protein 14 (Zip14) and upregulation of 7 genes expression including p53, superoxide dismutase 2 (Sod2), Heme oxygenase 1 (Hmox1), Caspase-3, Caspase-8, transferrin (Trf), and Bcl-2 in total 14 genes investigated in this study. In particular, Fos was down-regulated to 1.17–3.25 fold changes, whereas Homx1 and Caspase-3 were up-regulated from 0.73 to 1.64 times in the both NPs groups. Additionally, the residual genes used here had an opposite gene expression. For instance, mRNA levels of Metallothionein 1 (Mt1) and Metallothionein 2 (Mt2) were increased to approximately 0.48 fold change in AuNPs group, while their levels were declined to 2.17–2.64 times in AgNPs normalized to that in control group. Of note, the AgNPs treatment group exhibited the greater effect in most of gene expression compared to that in AuNPs group.The transcriptional levels of genes with functions associated to cell apoptosis, ion transport, and oxidative stress were further determined to understand the subtle differential damages of AgNPs and AuNPs in liver deposited highest amount of NPs in this study. As presented in Fig. 40. Zhang et al.11 demonstrated that 5, 10 and 45 mg/kg AgNPs caused a decrease in the body weight. Also, the liver and spleen mass increased while the thymus showed weight decrease with exposure to low doses of AgNPs (<6 mg/kg)28. However, the body weight and organ index were insignificantly affected by oral and inhalation exposures to both NPs42. Increases in brain weight and in liver weight were observed only at doses up to 300 mg/kg per day in a 28 d oral toxicity study20.We aimed to distinguish the biodistribution and toxicity differences between AgNPs and AuNPs by examining the body weight, organ index, organ distribution, hematology and biochemistry, histology, and gene expression profiles. First, we observed a mass decrease in spleen, brain and testis but no reduction of body weight after intravenous administration of 11.4–13.3 mg/kg AgNPs and/or AuNPs in mice. The body and organ weight proved to be susceptible to inorganic NPs intravenous exposuree.g. Kupffer cells in the liver and the macrophages and B cells in the spleen that are part of the MPS27. A relatively small amount of AuNPs or AgNPs were accumulated in the kidney, lungs, heart, stomach and intestines. AgNPs and AuNPs levels showed lower levels in the liver and small increments in the kidney and spleen at 28 dpi, indicating possible translocation of AgNPs and AuNPs in vivo. Of note, this translocation led to more accumulations in the testis and brain over time, implying that 3 ± 1.57 nm AgNPs and 6 nm AuNPs could effectively cross the blood-testis barrier (BTB) and the blood-brain barrier (BBB). The AgNPs with 28 oral administration was not cleared out of the brain and testis during a 4-month recovery period, which was consistent with our results, albeit over a shorter period of time14. Nevertheless, the total recovery ratios of administrated NPs were less than 50% in all groups, indicating that the residual amount of both NPs could be mainly stored in the carcass. Previous studies showed that AgNPs concentrations in the urine were extremely low compared with the concentrations in the feces43. NPs with sizes <5.5 nm exhibited rapid and efficient urinary excretion, while 10–20 nm NPs were trapped in the liver and eliminated in the feces44. Renal excretion of AgNPs and AuNPs with sizes of 12.22 ± 6.05 and 15.07 ± 6.68 nm respectively could not be the primary route of elimination. Furthermore, a small number of AgNPs and even fewer AuNPs were detected in the feces, suggesting that the AgNPs but not the AuNPs were excreted through the biliary pathway from the liver to the bile duct, intestine, and feces37.The majority of AuNPs and AgNPs were accumulated in the liver and spleen, which could due to uptake by the resident phagocytes 25; and also NPs with approximate HD of 10–20 nm had a similar elimination behavior in vivo44. Previous studies suggested that a complex protein corona is formed immediately when NPs were incubated in a biological fluid, and the surface coating controlled the composition and structure of the protein corona which was relevant to the pathophysiology in vivo47. In this study, the identical surface coating (amphiphilic polymer coating with a reactive group of carboxylic acid) used for both NPs was selected to exclude the impact of surface physicochemical properties during the formation of the dynamic protein corona. The difference in distribution and circulation of AgNPs and AuNPs in vivo, therefore, could be attributed to the intrinsic properties of the NPs that lead to the formation of diverse protein coronas on the surface of AgNPs and AuNPs, rather than the size of the NPs. Additionally, in model zebrafish embryos, Bar-Ilan et al.34 demonstrated that AgNPs induced more severe lethal and sub-lethal effects than AuNPs at the same concentrations and sizes, implying that the NPs chemical compositions play a vital role in toxicity induction. The intravenously administered AgNPs were stored primarily in the liver and spleen, which then released silver ions that migrated and accumulated in the kidney, lung, and brain30. More widely accumulated AgNPs in mice that were observed in our study were therefore probably due to the released silver ions that easily translocated into organs.The difference in HD of the AgNPs and AuNPs (12.22 ± 6.05 and 15.07 ± 6.68 nm) could show minimal effects on the distribution and toxicity of NPs because AuNPs with HD of 11 nm and 21 nm showed no significant differences in terms of accumulations in the liver, spleen, lung, and kidney as well as in terms of body clearance from feces and urine at 24 h after administration20, though no obvious injuries occurred in organs of present study. Caspase 8 and Caspase 9 as initiator caspases are activated by death-inducing tumor necrosis family receptors and cytochrome c, respectively, which are involved in immune and apoptosis signaling. Caspase-3 mediates mitochondrial targeted apoptotic signaling pathways which is an executor of apoptosis49. Activation of p53 is associated with apoptosis through regulation of Bcl-2 and Bax gene expression. Bcl-2 is a type of apoptosis inhibiting factor while Bax regulates the apoptosis activation factors such as cytochrome C, ratio of their expression level reflects the state of cell apoptosis. The transcription levels of these apoptosis markers revealed that gene expression was moderate regulated in both groups, while more effectively changed was observed in liver of mice treated with AgNPs. ROS is also considered to be a mechanism of genotoxicity for metal NPs, which could promote oxidative DNA damage22. Proteins of Sod1 and Sod2 are capable of scavenging free superoxide radicals and Homx1 is an ROS sensor which exhibited excellent anti-inflammatory and antioxidant properties49. Stable gene expression of Sod1 and Sod2 and small enhancement of Hmox1 expression indicated inefficient oxidative stress was induced in the liver of mice exposure to both NPs. Moreover, degradation of metal NPs in vivo could influence the ion transport of essential trace metal elements36. Trf is an iron transporter and Zip-14 is an iron and zinc transporter showed minimal effects in expression when mice exposure to NPs. However, Metallothionein (MT) is considered to be the essential indicator of toxicity caused by heavy metals and/or metal NPs as facilitating detoxification of metal and protection from oxygen free radicals49. Mt1 and Mt2 were significantly down-regulated in liver of mice treated with AgNPs as our previous study for iron oxide magnetic NPs36 referred to free silver ions could be released from AgNPs. Their expressions in AuNPs group showed an opposite trend with negligible upregulation manifested that AuNPs should be more inert in vivo.Evaluation of genotoxicity is an ideal tool to assess the biosafety at molecular level for metal NPs without induction of apparent histologic lesions. The hypothesis was that susceptive genes related to oxidative stress and apoptosis could be affected as proved in previous studiesMt1, Mt2, Fos, and Caspase-3  in livers of mice treated with both NPs on 28 dpi, while more effectively changed was observed in AgNPs group. As shown on Fig. in vivo should be conducted to distinguish the toxicity differences caused by the chemical composition of the NPs to gather valuable information for safety.In conclusion, the present study demonstrated that the AuNPs were mainly stored in the liver, whereas the AgNPs were widely stored in more organs including the heart, lungs, kidney, etc. The circulation of the NPs in the blood and excretions in the feces were also found to differ between the AgNPs and the AuNPs. The transcription levels of biomarkers of apoptosis, oxidative stress, and ion transport were not significantly altered except 36. The HD distributions of the AgNPs and AuNPs (n = 4) and zeta potential were measured by DLS using a Zetatrac Ultra 151 .Water soluble AgNPs and/or AuNPs with amphiphilic polymer coating and carboxylic acid as the reactive group used here were obtained from Ocean NanoTech, LLC . Two NPs sizes were confirmed with TEM prior to use. Briefly, NPs samples were prepared by dropping a sample onto an agar carbon-coated copper grid (400 meshes) and the solvent was evaporated. The TEM images were obtained at 50–100 K magnifications with a JEOL transmission electron microscope  operating at 100 kV as previously describedHealthy, 10–12 week old, adult male KunMing mice (30–35 g each) were purchased from the experimental animal center of Nanchang University, China. The animals were raised in an animal facility at 25 °C with a 12 h light/dark cycle; the animals were supplemented with food and water ad libitum. All procedures involving animals were approved by the Animal Care Review Committee , Nanchang University, Jiangxi, China, and were performed in accordance with the relevant guidelines and regulations of institutional animal care committee.28 and much less than the doses used in several oral exposure studies29. The weight of the mice and food consumption were recorded throughout the entire experimental period. Collection of the blood samples and isolation of various organs were as described in our earlier study36. In brief, blood samples were harvested from the five mice in each group on day 1 and 28 dpi and then the mice were sacrificed. The organ samples were collected and weighed for visceral index measurement. Two samples of the organs were isolated and a small portion of tissue was immediately fixed in 10% neutral buffered formalin for histological examination. The remaining portion from each organ (0.1–0.5 g) was stored at −20 °C for elemental analysis. The organ indexes were calculated using the following eq. . Exposures were carried out via tail-vein injection with 0.1 mL of 0.5 mg/mL AgNPs or AuNPs suspension or physiological saline (control) each time, 2 times a week, for 4 weeks. The injected dosage was approximately 11.4–13.3 mg NPs/kg body weight in total, which was similar with the doses used in relevant intravenous exposure studies3:HClO4 = 5:1) and were heated to 230 °C. The temperature was increased to 280 °C when the reaction reached equilibrium. The digested organ samples were diluted with Milli-Q water to 25 mL after removal from the heating block. For AgNPs treatment group, the digested subsamples were used to determine the silver concentrations with AAS  when the concentration of AgNPs was greater than 0.05 mg/L and ICP-MS  when the concentration of AgNPs was less than 0.05 mg/L. With the AuNPs treatment group, the digested subsamples were used to determine the gold concentrations with ICP-OES  when the concentration of AuNPs was greater than 0.05 mg/L and ICP-MS when the concentration of AuNPs was less than 0.05 mg/L. The concentration of AgNPs and AuNPs in the isolated organs from the control animals was determined using ICP-MS. All sample digestions and measurements were performed by using the standard protocol established by Center of Analysis and Testing of Nanchang University, Nanchang. The contents of AgNPs and AuNPs in the different tissues were calculated using the following eq.  and approximately 0.3 mL whole blood were evaluated for concentrations of AgNPs or AuNPs. The subsamples were dissolved in 12 mL digestion solution  was collected in potassium EDTA collection tube for hematology, and approximately 0.8 mL blood was centrifuged  to obtain at least 0.25 mL blood plasma for serum biochemistry study. The residual blood of each mouse was used to determine the AgNPs or AuNPs contents. Indicators of liver and kidney functions such as ALT, AST, TBIL, ALB, A/G, GGT, ALP, BUN, and UA were monitored. WBC, RBC, HB, MCH, PMN, LY, and PLT were also determined. The whole blood with anticoagulant and the serum were examined and followed the guidelines and regulations of the First Affiliated Hospital of Nanchang University, Nanchang, China.A small portion of each organ was fixed in 10% neutral buffered formalin. Subsequently, isolated tissues were embedded in paraffin blocks (previously melted at 58 °C) and frozen at 4 °C before 3–5 µm sections were cut and stained with hematoxylin and eosin (H&E) for histological examination. An Olympus optical microscope  was used to observe the stained slices.36. In brief, RNA was extracted and cDNA was synthesized using the AxyPrepTM Multisource Total RNA Miniprep Kit  and the Takara PrimeScriptTM RT reagent kit , respectively, according to the manufacturer’s protocol. The isolated total RNA was examined by agarose gel electrophoresis and quantified by using the NanoDrop 1000 spectrophotometer (Thermo scientific Inc.) with total RNA (1000 ng) for cDNA synthesis. Table −ΔΔCt method, with GADPH utilized as the reference gene for whole groups.Since highest deposition of both NPs in the liver which was selected to the representative organ to determine the sensitive gene expression. The total RNA extraction, cDNA synthesis, and Quantitative PCR analysis were performed as our previously studyAll the data were expressed as means ± standard deviations (n = 5). Comparison of results among the groups were carried out by one-way analysis of variance (ANOVA) and L.S.D. test using SPSS v16.0 ; *P < 0.05 and **P < 0.01 were considered significant statistically and extremely significant statistically, respectively.Supplementary Information


Bothrops atrox, common fer-de-lance viper, in the remote rainforest of eastern Ecuador and without access to definitive care for seven days. Antivenom was not administered; by the time of presentation to a hospital, he had suffered myonecrosis of his lower leg, which was treated with debridement and eventual skin graft. The ramifications of this long evacuation demonstrate the need for more accessible health services and educational outreach.This report describes a patient envenomated by a  Bothrops genus.B. asper is responsible for the majority of ophidic morbidity and mortality throughout Latin America. Studies have demonstrated that incidence and death rates  in Ecuador are consistent with neighboring rural Brazil and Colombia.6The estimated 400,000 snakebites that occur annually worldwide are a serious problem for the regions they affect, primarily in nations with lower income and resources.B. atrox. His associated symptoms included gingival bleeding, hematemesis, abdominal pain, oliguria, and shortness of breath. He denied fever, chest pain, or neurological changes.A 36-year-old Ecuadorian male was transferred from a rural health center to a district-level hospital with worsening right lower leg pain, edema, and skin discoloration following a snakebite in a remote area of the Amazon seven days prior. He stated that he was walking in the jungle barefoot when he sustained an unprovoked bite on his right ankle. He beheaded the snake and walked to a neighboring shaman who provided local herbs and prayer for one day. He required several days of remote travel by foot and canoe to arrive at a community where air evacuation could be arranged for the remainder of the 150 miles to the district hospital. He identified the snake as an “equis,” the common name in the area for the Triage vital signs were temperature 36.7 °C, blood pressure 100/60 mm Hg, heart rate 104 beats/min, respiratory rate 20 breaths/min, and SpO2 95%. Physical exam revealed the patient in no acute distress. His lower leg was edematous with areas of hyperpigmentation and ulceration extending to the dorsal foot. Labs showed a 12-minute whole blood clotting time , creatinine of 3.25 (0.5–1.2 mg/dL), urea of 174 (7 to 20 mg/dL), and a normocytic anemia with a hemoglobin of 5.3 (13.5–17.5 g/dL) and hematocrit of 14.1(43–52%). Ultrasound of the leg showed preserved blood flow and a fluctuant (4 cm × 1 cm) area in the anterior compartment; incision and drainage yielded 4mL of purulent fluid. Abdominal ultrasound revealed decreased renal corticomedullary differentiation suggestive of inflammation without obstruction.The patient was diagnosed with a viper bite complicated by an abscess of the anterior tibial compartment and grade 3 renal insufficiency. The patient received two units of packed RBCs and was started on intravenous fluids, erythropoietin, iron, folic acid, enalapril, oxacillin, and acetaminophen. Subsequently, the patient was transitioned to clindamycin and started on enoxaparin for venous thrombosis prophylaxis. His necrotic tissue required staged debridements in the operating room, resulting in a large wound with exposed tendons; the wound was treated with daily bandage changes and sugar application for one month prior to successful skin grafting . All carBothrops venom in rats showed an elimination half-life of 12 hours, with no circulating venom detected after seven days postenvenomation.1This case emphasizes the sequela of delayed definitive care in snakebites, which occurs far too commonly in rural areas of Ecuador. The risk of dying or developing serious complications from a snakebite is significantly higher (relative risk = 2.5) in those who do not receive antivenom in the first two hours after a snakebite.Snakebite treatment protocol exists at the hospital in which the man was treated; however, given that the bite occurred over seven days prior to presentation, deviation from the protocol occurred and the patient was treated according to protocol and expert opinion. As a result, the patient was treated primarily using the indicated supportive treatment. 8Hemotoxic effects such as this patient’s gingival bleeding and anemia are common and mediated by a variety of venom components including zinc-dependent metaloproteinases.3Our patient was geographically isolated and spent days receiving shamanistic care. These are common barriers for those living in remote rainforest communities that prevent prompt definitive care of snakebites. Approximately half of those bitten in Latin American countries will be treated first with traditional methods such as plants, chemical products, physical methods, or prayer.


The objective was to investigate the effective and safe range of paramedian CDH by percutaneous posterior full-endoscopy cervical intervertebral disc nucleus pulposus resection (PPFECD) to provide a reference for indications and patient selection. Sixteen patients with CDH satisfied the inclusion criteria. Before surgery the patients underwent cervical spine MRI, and the distance between the dural sac and herniated disc was measured. An assessment was performed by MRI immediately after surgery, measuring the distance between dural sac and medial border of discectomy (DSMD). The preoperative average distance between the dural sac and peak of the herniated disc (DSPHD) was 3.87 ± 1.32 mm; preoperative average distance between dural sac and medial border of herniated disc (DSMHD) was 6.91 ± 1.21 mm and an average distance of postoperative DSMD was 5.41 ± 1.40 mm. Postoperative VAS of neck and shoulder pain was significantly decreased but JOA was significantly increased in each time point compared with preoperative ones. In summary, the effective range of PPFECD to treat paramedian CDH was 5.41 ± 1.40 mm, indicating that DSMHD and DSPHD were within 6.91 ± 1.21 mm and 3.87 ± 1.32 mm, respectively. PPFECD surgery is, therefore, a safe and effective treatment option for patients with partial paramedian cervical disc herniation. Cervical disc herniation (CDH) is one of the most common degenerative spinal disorders and is characterized by upper extremity pain and neurological deficits. Depending on the site of the intraspinal disc herniation, CDH can be divided into three categories: median, paramedian, and lateral herniations . There aIn an effort to reduce such surgery-related complications, there has been a push to develop advanced endoscopic techniques, of which full-endoscopic cervical discectomy (FECD) has been extensively used. FECD falls into two main categories: anterior (AFECD) and posterior (PFECD). AFECD is mostly used for patients with median CDH, which can effectively resect a protruded intervertebral disc to decompress the spinal cord. However, this is a risky approach given the proximity of major blood vessels and nerves . FurtherSixteen patients  were recruited for this study. The patients presented with paramedian CDH, as defined by a paramedian herniation that pressed the spinal cord unilaterally and deformed it into a comma shape, pressing the spinal cord and nerve root and then generating myeloradiculopathy symptom . All theThe inclusion criteria were as follows: (1) having received failed conservative treatment lasting for more than 4 weeks or symptoms deteriorating to the extent of becoming unbearable; (2) neurological symptoms (myelopathy and/or myeloradiculopathy) consistent with the preoperative magnetic resonance image; and (3) single-level paramedian disc herniation. The exclusion criteria were as follows: (1) clear segmental instabilities or deformities; (2) cervical intervertebral disc with calcification; (3) isolated neck pain for which the cause could not be determined by magnetic resonance imaging (MRI); (4) foraminal stenosis without disc herniation; (5) multiple-level disc herniation; (6) previous surgery at the same segment; and (7) a suspected infection or tumor in the cervical spine.The examinations were performed by two surgeons with experience in this technique. The vertical distance between the lateral border of the dural sac and peak of the herniated disc (DSPHD) and the distance between the lateral border of the dural sac and the intersection of the dural sac and medial border of the herniated disc (DSMHD) were recorded by two independent doctors from MRI images . The finOperations were performed under general anesthesia with the patients placed in a prone position with heightening at the chest to keep the neck flexed. The patients' shoulders were immobilized with tape and the arms placed caudally on the body with gentle tension to aid the fluoroscopic visualization of cervical levels. The line of spinal joints was marked using posterior-anterior radiography guidance, whereas the operations were guided by lateral radiography. Once the location of the cervical segment had been accurately determined, a skin incision was made and a dilator with a 6.9 mm outer diameter bluntly inserted into the facet joint. The operation sheath was inserted via the beveled opening of the operation performed under visual control and the site was irrigated continuously with 0.9% saline solution. The facet joint was completely exposed and grinded with a high-speed grinding drill. The lateral ligamentum flavum was resected to expand the intervertebral foramen to allow the endoscope to penetrate into the spinal canal, after which the herniated disc tissue was resected. The nucleus pulposus of the cervical intervertebral disc was ablated by radiofrequency (RF) ablation; the mobilization of the nerves root was repeatedly checked . FinallyAll patients were followed up 3, 28, 90, and 180 days after surgery, each patient receiving a questionnaire by mail four working days ahead of their attendance at the clinic. The follow-up examinations were conducted by two physicians, neither of whom had been involved in the operations. Besides general parameters, other relevant information was collected using the following evaluations: the modified Macnab criteria were used to evaluate the postoperative outcomes, whereas VAS and JOA scores were recorded at the final follow-up visit , 18. MRIt-test. In all analyses, a probability < 0.05 was considered significant. Results were presented as a mean ± standard deviation.Statistical analysis was performed using the Statistical Package for the Social Sciences . The Tamhane test and Dunnett test were applied to compare pre- and postoperative VAS and JOA scores at various times. The differences between pre- and postoperative distance measurements were analyzed using a paired sample None of the patients experienced any preoperative or postoperative complications, such as postoperative bleeding, injury to the nerve or dura, damage to the spinal cord with hemi-/paraparesis or paralysis of the upper extremities. There were not any complications from infection, spondylodiscitis, or thrombosis. Deterioration of existing symptoms was not observed in any of the patients.P < 0.05). The decrease in VAS, when comparing postsurgery day 3 with days 28, 90, and 180, were also statistically significant (P < 0.05). However, there was no significant difference between VAS scores recorded at days 28, 90, and 180 (P > 0.05)  .P < 0.05). The difference in JOA score at day 3, when compared with JOA scores at days 28, 90, and 180, was significant (P < 0.05), whereas the differences amongst the JOA scores at days 28, 90, and 180 failed to reach significance (P > 0.05)  . ApplyinSurgical intervention represents the most efficacious clinical alternative for CDH cases that fail to respond to conservative treatments. An operation by a surgeon can effectively relieve the spinal cord or nerve root compression and promote the recovery of its function, thus achieving a significant improvement in clinical symptoms –21. In rWith percutaneous posterior endoscopic cervical intervertebral disc nucleus pulposus resections, the inner margin of the articular process is grinded with an abrasive drill to open a hole into the spinal canal. This greatly reduces damage to posterior ligaments, muscles, and bone, while retaining maximal biomechanical stability of the cervical vertebrae. A follow-up study of 87 patients found that two years after receiving percutaneous PFECD (PPFECD) operations, 87.4% of the patients reported no recurrence of neck or shoulder pain, and only 9.2% experienced occasional pain. Although the decompression results of PPFECD were similar to conventional ACDF, the operation-related traumatization was reduced . PPFECD Previously, PPFECD has been applied to CDH patients whose symptoms included radiculopathy with upper extremity numbness and pain . Since tWe selected the edge of dural sac as a position marker for primary reasons: (i) it has a clear boundary which makes it a good point of reference and (ii) the dural sac is rarely affected by the surgery so that risk of damage to the spinal cord is negligible. The removal of the cervical intervertebral disc protruding from inside the dural sac can increase the risk of spinal cord injury, due to stretching of the sac. Consequently, one of the key objectives of this study was to explore the range of resection possible with the PPFECD technique on cervical intervertebral disc herniating from inside the dural sac.In the postoperation follow-up, we found that the distance between the edge of the dural sac and the inside edge of the intervertebral disc was significantly smaller than between the edge of the dural sac and the inside edge of the herniated disc. In other words, the resected amount of actual intervertebral disc tissues was less than that of the preoperative measurements. A possible explanation for this could be that the nucleus pulposus, not otherwise protruding from the disc, was RF ablated, causing a decrease in the pressure inside the intervertebral disc thereby retracting the remaining herniated disc. Postoperative symptoms of all patients were improved at all cervical levels, and cervical MRIs showed no evidence of protrusions compressing the spinal cord or nerves in any of the patient. At the final 6-month follow-up, there were no reported complications, such as spinal cord injury or dural sac rupture. Based on the analysis of all the experimental data, we propose that the application of PPFECD for cervical disc herniation up to 6.91 ± 1.21 mm and peak of herniated disc up to 3.87 ± 1.32 mm is safe and effective and that the safe resection range of cervical intervertebral disc using PPFECD is up to 5.41 ± 1.40 mm within the border of the dural sac.In our study, all patients recovered fully from the operation, experiencing no serious or even minor complications and, most importantly, preoperative symptoms of pain, numbness, and fatigue were completely relieved at the six-month endpoint of the study. There were no reported symptoms of spinal cord injury, such as sensory disturbance, muscle weakness, pathological reflex, or defecation incontinence. PPFECD offers advantages such as smaller incisions, less tissue damage, and adequate nerve root decompression which result in faster postoperative recovery, with fewer complications, shorter hospital stays, and, therefore, reduced cost. These are benefits that point to PPFECD as a safe and effective surgical procedure for treating cervical disc herniation. With a more in-depth understanding of spinal anatomy and refinement of surgical techniques, the indication and application scope of PPFECD could be expanded even further, thus exploring new space for the treatment of paramedian type CDH.In this study, we have found that the effective range was 5.41 ± 1.40 mm from the border of the dural sac for percutaneous posterior full-endoscopic cervical disc herniation discectomy treated paramedian cervical disc. For patients with partial paramedian cervical disc herniation was up to 6.91 ± 1.21 mm and peak of herniated disc up to 3.87 ± 1.32 mm within the lateral border of dural sac. PPFECD surgery is a safe and effective treatment option.


Sotos syndrome (SoS) is characterized by overgrowth of prenatal onset, learning disability, and characteristic facial appearance; it is usually due to haploinsufficiency of NSD1 gene at chromosome 5q35. An Italian child was born at 37 weeks of gestation  showing cryptorchidism on the right side, hypertelorism, dolichocephaly, broad and prominent forehead, and narrow jaw; the pregnancy was worsened by maternal preeclampsia and gestational diabetes, and his mother had a previous history of four early miscarriages. The patient showed neonatal jaundice, hypotonia, feeding difficulties, frequent vomiting, and gastroesophageal reflux. After the age of 6 months, his weight, length, and head circumference were above the 97th centile; psychomotor development was delayed. At the age of 9 years, the patient showed also joint laxity and scoliosis. DNA sequence analysis of NSD1 gene detected a novel heterozygous mutation  in exon 2. The same mutant allele was also found in the mother and in the maternal grandfather of the proband; both the mother and the maternal grandfather of the proband showed isolated overgrowth with height above the 97th centile in absence of other features of SoS. At present 23 familial cases of SoS have been described (two cases with mutation in exon 2 of NSD1 gene); no familial cases of SoS with mutation of NSD1 gene and isolated overgrowth have been reported. Probably, point mutations of NSD1 gene, and particularly mutations between exon 20 and exon 23, are not likely to affect reproductive fitness. Epigenetic mechanisms and intrauterine environment may influence phenotypes, therefore genetic tests are not useful to predict the phenotype but they are indispensable for the diagnosis of SoS. This is the first Italian familial case of SoS with genetic confirmation and the third report in which a missense mutation of NSD1 gene is found in three generations of the same family. Sotos syndrome (SoS) was first described by Juan Sotos in 1964; it is a rare genetic disorder whose prevalence is estimated to be 1:14,000 live births .Sotos syndrome is characterized by overgrowth of prenatal onset, learning disability, and characteristic facial appearance: it has been estimated that at least 90% of patients with SoS present these features . The heaLess frequently, patients with SoS present with behavioral problems such as autistic spectrum disorders, phobias, and aggression . CardiacLess than 15% of individuals with SoS present with cataract, conductive hearing loss, craniosynostosis, vertebral anomalies, talipes equinovarus, hemihypertrophy, gastroesophageal reflux, hypothyroidism, and cryptorchidism . Tumors de novo” t translocation in a child with SoS led to the discovery that SoS is usually due to haploinsufficiency of the NSD1 gene that encodes a histone methyltransferase (the nuclear receptor SET domain-containing protein 1); the NSD1 gene is located at chromosome 5q35, and its haploinsufficiency is caused by various mechanisms  q35;q24. transloc)q35;q24. transloc)q35;q24. translocNSD1 and the GH/IGF1 axis is still unclear , his length was 50 cm (75th centile), and his head circumference was 36 cm (97th centile). Apgar score was 8 at 5 min. Physical examination performed at birth revealed cryptorchidism on the right side, hypertelorism, dolichocephalic head with broad and prominent forehead, and narrow jaw. Blood glucose monitoring in the 24 h following the birth was normal; 48 h after birth the newborn showed neonatal jaundice that required phototherapy. Cardiac and abdominal ultrasound examinations were normal; cerebral ultrasound study showed a mild periventricular hyperechogenicity.The patient showed hypotonia and severe feeding difficulties since he was 3 weeks old; at the age of 1 month, gastroesophageal reflux was diagnosed, and frequent vomiting was present since he was 6 weeks old. At the age of 7 weeks, the child had an episode of Brief Resolved Unexplained Event with cyanosis, absent breathing, marked hypotonia, and unresponsiveness. Later, he showed some episodes of apnea with perioral cyanosis. For these reasons, the patient underwent cardiorespiratory monitoring and artificial feeding with nasogastric tube for 10 days.The weight gain of the patient was poor until the age of 6 months because of hypotonia, feeding difficulties, gastroesophageal reflux, and frequent vomiting. Later, he showed excessive growth, and his weight, height, and head circumference were above the 97th centile for age and sex. Psychomotor development was delayed: the child held up his head at 5 months, rolled over at 7 months, walked alone at 24 months, spoke a first word at 30 months and simple phrases at 4 years. At 4 years 6 months, he showed advanced bone age corresponding to 6 years.At the age of 9 years, the patient weighed 51.4 kg (>97th centile); he was 148.1 cm tall (>97th centile), and his head circumference was 60 cm (>97th centile). Physical examination revealed dolichocephaly, broad and prominent forehead, downslanting palpebral fissures, narrow jaw, hypertelorism, increased limb length, joint laxity, and scoliosis; his right testicle was smaller than his left one. Intellectual impairment and difficulties with speech and language were noted. Sideropenic anemia was demonstrated at blood examination.Clinical findings, after a differential diagnosis with other overgrowth syndromes Table , supportGenomic DNA was extracted from peripheral blood lymphocytes using EZ1 DNA kit and BioRobot EZ1 . All coding exons (exons 2–23) and exon–intron boundaries of the NSD1 gene  were amplified by polymerase chain reaction (PCR) (primers sequences and PCR conditions are available on request). PCR products were purified with QIAquick PCR purification kit  and directly sequenced with Big Dye Terminator Sequencing Kit version 3.1 , with the same primers used for the amplification. Finally, samples were run on 3130xl Genetic Analyzer  and analyzed with Sequencing Analysis Software .In silico analysis by PolyPhen-2 predicted it as probably damaging; analysis with Mutation Taster predicted the mutation as disease causing. The residue is conserved among different species.DNA sequence analysis of the NSD1 gene detected a novel heterozygous mutation: c.521T>A  in exon 2. The same mutant allele was also found in the mother and in the maternal grandfather of the proband. The mother was the only child of unrelated parents. She was 45 years old at diagnosis with height of 181.6 cm (>97th centile); her weight and head circumference were normal. She had neither facial appearance suggestive of SoS nor intellectual impairment; she graduated at University and worked as employee. She was born as appropriate-for-date, no neonatal complications were observed, and her psychomotor development was normal. She suffered from Hashimoto thyroiditis and had a previous history of four early miscarriages.The maternal grandfather of the patient was 79 years old at diagnosis with height of 195.4 cm (>97th centile); his weight, head circumference, and facial appearance were normal. He was born as appropriate-for-date, no neonatal complications were observed, and his psychomotor development was normal. He graduated at senior high school with good results and had worked as employee.At present, the child undergoes periodic clinical examination and investigations because of the increased risk of tumors and other complications; orthopedic follow-up for scoliosis is also performed. The child participates in a regular class with support, and his parents undergo periodic psychological counseling.Written informed consent for the publication of the present case report was obtained from both parents of the proband and his maternal grandfather.We hypothesized our patient should suffer from SoS because of his history of overgrowth, his facial appearance, and the presence of many other suggestive findings Table  2, 5–7), 5–72, 5In our family, the same mutation in exon 2 of the NSD1 gene was present in the proband, in his mother and in his maternal grandfather; all the three patients showed overgrowth. In addition to overgrowth, the proband showed most of the features of SoS. At present 23 families with at least two family members affected by SoS have been identified as carrier of mutations of NSD1 gene associated with overgrowth and other clinical findings of SoS Table –18. Ho¨gNSD1-positive patients identifying mutations in 11, all of whom showed clinical findings of SoS; in other words, they have not seen a case of incomplete penetrance for a confirmed pathogenic NSD1 gene mutation  showed the mutation of NSD1 gene associated with isolated overgrowth; this finding has never been reported previously among familial cases of SoS. Tatton-Brown et al. screened more than 300 parents of mutation . On the ergrowth .According to Table NSD1, and intrinsic variability in the regulation of downstream targets of NSD1 may be responsible of a different expression profile of NSD1 gene; the resulting tissue-specific gene expression profile may possibly influence phenotypes of SoS , possibly inherited from their father, had already been described previously but genetic tests for SoS were not available at that time, therefore our family represents the first Italian familial case of SoS with genetic confirmation . In addis of SoS . AccordiIn conclusion, we identified an Italian family with a novel missense mutation in exon 2 of NSD1 gene; each member carried the same molecular abnormality with variable phenotypes and severity, from isolated overgrowth to classical SoS. The present family case report will contribute to the understanding of the correlation between genotype and phenotype in familial SoS.Written informed consent for the publication of the present case report was obtained from both parents of the proband and his maternal grandfather.GL, FM, AG, FL, SL, and PG met the patient to each visit and wrote the present case report; AM performed the molecular analysis of NSD1 gene.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.