Towards developing a polymeric matrix characterized by high strength to cost ratio, polypropylene (PP) was hybridized with low-cost particulate snail shell (PSS) and kenaf fiber (KF) via compression moulding at 180 °C and 0.2 MPa. The developed composites were grouped into three and labeled as mix 2, 4, and 10. Each group entailed the blend of 5, 10, 20, and 30 wt% KF with 2, 4, 10 wt% PSS respectively. From the results, it is observed that the hardness value was enhanced by the blend of 5 to 30 wt% KF and 2, 4, and 10 wt% PSS. However, 2 wt% PSS mix with 5 to 30 wt% KF resulted in progressive improvement in impact, compressive, flexural, and tensile strengths values. The 4 wt% PSS yielded consecutive increase in impact, compressive and flexural strength when combined with 5 and 10 wt% KF. However, it was observed that subsequent addition of 20 and 30 wt% KF led to a marginal reduction in the strength values. The tensile strength attained optimum value when 4 wt% PSS was commixed with 30 wt% KF. Conversely, the combinations of 10 wt% PSS with 5, 10, 20, and 30 wt% KF had no significant improvement to the mechanical properties of PSS/KF-bio-PP composite (except for hardness) siring strength decrease. Taguchi optimization revealed that the collage of 4 wt% PSS and 10 wt% KF presented optimum mix for hybrid bio-PP composite. Polymer, on the other hand, is light in terms of weight, possesses a high strength-to-weight ratio, low cost, and has resistance to corrosion3. Nowadays, polymer has found successful engagement in most engineering applications as substitutes to metals because of cost implication and corrosion resistance4. Engineering polymers are employed in biomedical and biomimetics, aerospace, automobile, electricity and electronics, structures, clothing, and material development6. Polymeric materials are further classified into thermoplastics, thermosets, and elastomers. Thermoplastics like polypropylene, polyethylene, and polyvinyl chloride have high demand based on their everyday use. Polypropylene (PP) for instance, is applied in packaging, automobile, textile, medical and other forms of application8. Being an engineering material, it has good chemical and corrosion resistance, it is tough, and possesses good fatigue and heat resistance9. According to Maddah10, energy-saving policy in automobile manufacturing necessitates the use of lightweight engineering materials for fuel optimization. Based on this and owing to the lighter weight of PP when compared with most polymeric materials, PP is being used for different parts of the vehicle. Some of the areas of use in cars are dashboards, batteries, AC ducts, doors, and quarter panels13. Also, thermoplastic olefin produced from PP is utilized in car bumpers, air drums, and rocket panels15. In aerospace, PP is employed for sandwich composite panels while in structures, it has application as fibers for reinforcing concrete17. For better performance in service, base polymeric matrix undergoes structural alteration and modification via different processes one of which is reinforcement with different materials; infusing synthetic and agro by-products in the matrix. Agro-by-product reinforcement of polymer matrix in form of bio fillers and bio fibers are encouraged based on their low cost and reuse. The agro-based products cause some modification at the microstructural level bringing about enhanced property of base material. Authors Onuegbu and Igwe18 investigated the effect of snail shell powder on the properties of PP. The snail shell powder was sieved to 15 µm, 30 µm, and 42 µm after which they were added at varying proportions of 10, 20, 30, and 40% respectively. The mechanical properties were assessed and the result revealed progressive appreciation in tensile, impact, and flexural strengths. Analysis of the results showed a higher improvement of properties with reduced particle sizes. Optimum mechanical properties were attained at 40% of the powder for all particle sizes considered with 15 µm having the highest performance. Studies from Onuegbu and Nwanonenyi19 examined the mechanical properties of PP by adding pulverized groundnut husk at 2, 4, and 6%. Particle sizes were varied at 0.2, 0.4, 0.6, 0.8, and 1 mm. The result showed enhancement in tensile, flexural, and impact strengths on account of adding up to 6 wt% of the powder. From the outcome, the inclusion of filler contributed to the property improvement of the PP matrix. On the other hand, coconut shell particles have been reported as a strengthener in the production of polymer matrix composites20. Varying weight percent of coconut shell particles at 10, 20, 30, and 40 wt% were utilized in the production of polymer-based composites. The results revealed that irrespective of size, an increasing proportion of the particle improved the yield, tensile and flexural strengths even as hardness was enhanced20. In recent times, cardanol was used with untreated and NaOH treated coir particles at different sizes of 25, 50, and 75 µm 21. Their studies revealed an enhancement in tensile, flexural, and impact strengths respectively. Further observation revealed that 25 µm treated coir particles performed optimally. Equally, 3, 6, 9, 12, and 15 wt% snail shell powder were observed to improve tensile strength in recycled waste plastic developed for automobile application12. Similarly, compressive, impact, and flexural strengths, and hardness were enhanced by the blend of 10, 20, 30, 40, and 50 wt% of carbonized eggshell filler22. Researchers also investigated the influence of agro-fiber as reinforcement in polymers reporting various outcomes. Natural fibers like sisal, banana, and coir were employed in reinforcing epoxy resin in the literature23. Tensile strengths, flexural strengths, and hardness of the epoxy composites were enhanced by the fibers. Another common fiber is bagasse24. Authors Akinwande et al.25 examined the effect of proportion and length of coir fiber in PP towards property enhancement for automobile application. Experimental results depicted that the inclusion of fiber at 5 to 20% played a major role in improving tensile and flexural properties. Impact resistance was enhanced up to 25 wt% of fiber while hardness showed progressive improvement with coir fiber fraction of 5 to 30 wt%. Optimization engaged in via response surface methodology denoted fiber length 34.85 mm at 25.76% proportion is most desirable. Kenaf/polypropylene composite was developed by authors Lee et al.26. As observed, 10, 15, 20, and 25 wt% kenaf fiber-enhanced tensile and flexural properties over neat PP depicting the fact that kenaf fibers in PP ensued positive modification of mechanical properties for engineering application. From the findings of Ramli et al.27, PP was reinforced with oil palm fiber matrix. From the outcome, 40 wt% fiber reinforced PP showed better properties than control PP matrix with regards to flexural strength, tensile and flexural moduli. Jute fiber employed by Kabir et al.28, led to the enhancement of tensile and flexural properties. From these literatures, it’s clear that agro-by products in terms of fiber, play major role in the improvement of mechanical properties of developed polymeric composites. Additionally, hybrid polymer composites of fiber and particulate fillers blend had been a subject of investigation for years; as investigated by Balaji et al.29 which entailed a study on bagasse fiber/coconut shell hybrid biocomposites. On the other hand, hybridized polypropylene with coir fiber and yam peel particulate as reported by Adediran et al.30, showed an improvement in impact strength as well as tensile and flexural properties. In Adeosun et al.’s work31, hybrid polyester composite with the blend of coconut and snail shell particulate in unsaturated polyester was reported. By contrast, coconut shell particulate outperformed snail shell particulate especially tensile strength and Brinell hardness, while snail shell outperformed coconut shell with regards to bending and impact strengths. Generally, optimum results were obtained with portions between 10 and 20 wt% for the two particulates proving that dual particulate contributes to property enhancement of polymers. It is well known that, agro-fibers and particulate fillers have a promising future as reinforcement in polymers. This study, therefore, involved property evaluation of polypropylene matrix reinforced with kenaf fiber and particulate snail shell. The choice of polypropylene is on the dint of low cost and its availability in the Nigerian market. Improving low-cost PPmatrix with agro-byproduct can lead to the development of useful engineering products which are affordable. In addition to the property evaluation, statistical analysis was carried out on obtained results to derive an optimum combination of kenaf fiber and particulate snail shell.Fundamental engineering materials are in most cases classified as metals, ceramics, and polymers. These classes of material find application in various aspect of engineering and the choice are based on their properties. The selection of one class of material over another is on account of comparative advantage in terms of property and cost. Metals are characterized by high strength, ductility, and closed packed structures32 where surface treatment of kenaf fiber with 0.8% achieved optimum performance. Treated fiber  shell, and virgin polypropylene (PP) pellet. Kenaf fiber and snail shell were procured from a nearby farm in Omu-Aran, Kwara State, Nigeria while stearic acid and virgin PP pellet was procured from a merchant in Omu Aran, Kwara State. Procured kenaf fiber was washed in warm water to removed attached impurities and sundried for 2 days. Afterwards, the fiber was cut to 25 cm length and treated with 0.8% stearic acid according to procedures followed by Salman et al.ber Fig. a, was waber Fig. b, were cSurface treatment of kenaf fiber revealed a rough surface of the fiber occasioned on account of the removal of wax and impurities. The process also led to a reduction in lignin and hemicellulose content Table , thereby33 in which mechanical property optimization of low-density polyethylene matrix was realized by the incorporation of 2 to 10 wt% snail shell powder.The development process involved blending of particulate snail shell (PSS) and kenaf fiber (KF) in polypropylene (PP) matrix at varying proportions using waring laboratory blender (8011EG). The control mix was produced as pure polypropylene (PP) with no KF and PSS admix. Three group mixes of samples were considered and are labeled as mix 2 composites for the first group, mix 4 composites for the second group and mix 10 composites for the third group. Group labeled mix 2 composites entailed the blend of 0, 5, 10, 20, and 30% KF by weight of PP with 2 wt. PSS. Similarly, groups labeled mix 4 composites consist of the blend of 4 wt% PSS and 0, 5, 10, 20, and 30% KF and mix 10 composites contained the blend of the same proportion of KF and 10% constant PSS weight fraction. 2, 4, and 10 wt% PSS were considered based on conclusion made by Chris-Okafor et al.3) and 63.5 × 12.7 × 2.5 (mm3) according to Adediran et al.34. Samples for compressive strength were produced in moulds 40 mm diameter and 80 mm length. Wax was robbed in the mould cavity while the top and base of the mould were covered with Teflon sheets before compression. Samples were left to cure for 24 h sequel to careful removal from moulds and cleaning.Compression moulding machine (NG-BU-P11) operated at a temperature of 180 °C and pressure of 0.2 kPa for 10 min on each sample was employed. 180 °C was adopted because temperature trials above 180 °C produced burnt samples and below produced less compacted samples. Dump bell shape mould 150 mm length and 3 mm thickness was used in the preparation of samples for the tensile test. Samples for flexural and impact were prepared using a mould of dimension 150 × 50 × 3 (mm35. The pendulum of the tester was set at 165° to fracture samples and impact energy to fracture was measured. An average of three samples was recorded for each mix proportion.Izod impact strength was appraised by subjecting notched samples (V-shaped) to impact employing Hounsfield balance impact tester (3915) in line with ASTM D 256-1036 applying a load of 100 N at a crosshead speed of 5 mm/min. Flexural samples were subjected to a three-point bending load of 1 kN. A crosshead speed of 10 mm/min was adapted and a span length of 100 mm was maintained for all samples. The test was done in accordance with the ASTM D 790–1737 procedure. An average of three samples were noted for each mix proportion.A universal testing machine (Instron 3369 series) was used in the test in line with ASTM D 3039 M-1738 employing shore Durometer apparatus. The samples were indented five times on the surface and the average value was obtained.The hardness of the developed composite was probed as par ASTM D 2240-15e139. Samples were subjected to compressive load at a strain rate of 0.1 s−1 and piston speed of 0.25 m/s. An average of three samples were recorded for each mix proportion.Quasi-static compression was examined on cylindrical samples produced as prescribed by ASTM D 695-15Microstructural analysis was carried out with the use of High magnification fluorescence microscope (E-807Y) operated at 5.0 kV and magnification of × 5000.Figure 29 and Anuar et al.46. In their study, the inclusion of kenaf fiber at 5 to 20 wt% in maleic anhydride grafted polypropylene led to enhancement of strength of composites. Additionally, kenaf fiber of 10, 20, 30, and 40 wt% was observed to boast composites strength in Salman et al.32, though contrary observation was reported in Kim and Cho47 where 10, 20, and 30 wt% kenaf fiber manifested a progressive decrease in strength. Figure Figure 29 portrayed an uptrend in hardness with increasing banana fiber loading of 5, 10, 15, and 20 wt% in epoxy matrix; corroborating the outcome of this study.As observed in Fig. 41. Similarly, Durowaye et al.42 corroborated the findings of this study as date palm fiber loading showed improvement of hardness in polyester composite developed in their investigation. Findings from Swain and Biswas43 and Chelliah44 linked the improvement in hardness to the cross-link of fiber with the polymer chains resulting in a complex network.From the result obtained, the interaction between the KF and PSS led to the enhancement of hardness. It was reported that Shore D hardness showed an enhancement as fiber content increased from 0 to 20 wt%45 which explicated enhancement of hardness of low-density polyethylene matrix by the reason of adding periwinkle shell particles in the dosage of 2 to 16 wt%.Present study revealed infusion of 5, 10, 20, and 30 wt% KF in the PP matrix depicted 6.0, 9.0, 11.9, and 14.9% uptrend in the value with respect to the pure PP (control sample). This delineates the fact that inclusion of kenaf fiber with PSS in polypropylene matrix ensues improvement in hardness. In the case of mix 4 composites (4 wt% PSS) which involved commix of 4 wt% PSS at the constant amount and varying KF dosage of 5, 10, 20, and 30 wt%, it is revealed that increasing KF portion resulted in the progressive rise in values. As highlighted in Fig. Figure Figure For mix 10 composites which were doped with 10 wt% fixed proportion of PSS, it is evident from Fig. 48, 44.4% optimum enhancement of impact strength was observed with the use of chitosan microparticles (3.5 µm average diameter) whereas in present study, optimum enhancement of 54.4% enhancement was realized for the same strength. Likewise, optimum impact strength enhancement attained in present study is superior to 16.7% improvement realized in Husseinsyah et al.49 when 10 wt% chitosan filler was added to polypropylene matrix. Comparative result of Amri et al.50 reflected 20% optimum enhancement in impact strength of polypropylene matrix when 10 wt% chitosan was added, a value lower than optimum improvement realized in this study.With respect to the findings from Daramola et al.51, revealed that the mix of banana fiber in epoxy at 5 and 10% vol. fraction fortified the epoxy composites containing teak wood dust. It was noted that 5 and 10% banana fiber yielded enhancement of 7 and 14.9% in compressive strength at 10% teak wood dust. According to the study, 20% fixed teak wood dust combining with 5 and 10% banana fiber, kindled 5% reduction in and 13.1% increase in compressive strength respectively. Similarly, study of Adediran et al.34, noted that an increase in dosage of bamboo fiber resulted in an insignificant contribution to the compressive strength while particulate distribution in matrix unfurled significant effect on the strength.Few studies carried out on compressive strength of polymer composites has shown compelling outcomes. The report from Dash3 presence in PSS which contributed to strength value. Also, the ensuing close packing of particles and fiber interlock with polymer chains resulted in cohesive interaction between particles and fiber, engendering even stress propagation as obtained in Fig. In this study, Fig. 54. Findings from Sushanta55 revealed progressive enhancement of flexural strength of PP matrix reinforced with banana/glass fibers corroborating the observations reported in the present study. From Mir et al.’s study, kenaf fiber inclusion (KF) resulted in strength enhancement for KF in the proportion of 5, 10, 20, and 30 wt%.Natural fibers have proved to be good agents in improving the flexural properties of polymers41. It is evident that the evaluation of mix 4 composites (4 wt% PSS) displayed enhancement in flexural strength on the infusion of 5 and 10 wt% KF. In contrast, subsequent addition of 20 and 30 wt% KF amounted to 7.8 and 16.2% decline relative to 10 wt% KF value. In comparison, mix 4 composite samples blended with 4 wt% (PSS) performed better than mix 2 counterparts (2 wt% PSS) when 5 and 10 wt% KF was infused. This is based on enhanced interparticle cohesion owing to closed packing afforded by particulate presence.In the present investigation, the presence of KF in mix 2 composites reflected a consecutive rise in flexural strength with fiber loading which occurred on the dint of fiber-matrix chain network formed, coupled with enhanced adhesion between KF and PP matrix Fig. b. These 56. It was reported that incorporation of 0, 5, 10, 15, 20, and 25 wt% peanut husk powder in low-density polyethylene resulted in the progressive rise in flexural strength with 25 wt% of the powder kindling up to 63.3% enhancement. Similarly, findings of Asuke et al.57 revealed the same trend when 0, 5, 10, 15, 20, and 25 wt% of carbonized and uncarbonized fish bone powder was infused into polypropylene matrix. At 25 wt%, enhancement of 53.8 and 35. 9% were realized for both carbonized and uncarbonized respectively. These reports are in tandem with the findings showcased in the present study.Particulate addition has been reported in some studies to enhance flexural strength as noted by Obasi58 studied the effect of natural hydroxyapatite on mechanical performance of poly (methyl methacrylate) biocomposites. Hydroxyapatite content from 5 to 20 wt% ensued progressive reduction in flexural strength and strain contrary to observation realized in this study.As noted in Fig. 59, Haque et al.60, Haque and Islam61 revealed that fiber inclusion in polymer matrix up to 20 wt% ensued enhancement of tensile strength. Findings from Balaji et al.29 achieved optimum tensile strength at 15 wt% banana fiber an increase of 65 and 68.9% over neat epoxy, for 10 mm and 20 mm length of the fiber respectively.The result of tensile strength against KF/PSS is as presented in Fig. Meanwhile, kenaf fiber employed in present study had an optimum tensile at 20 wt%, an enhancement of 72.9% over neat PP. It is noteworthy that 30 wt% KF had a slight reduction of 3.2% in strength compared with value attained at 20 wt% KF. The reduction is fraternized by possible reduced stretching of fibers owing to frictional interaction between the two input materials. 5, 10, 20, and 30 wt% KF resulted in 11.0, 36.4, 72.9, and 62.3%.62 in which 5, 10, 15, 20, and 25 wt% led to the progressive decrease in strength associated with possible stress concentration at particulate/matrix boundary. By contrast, the 10 wt% chitosan microparticle from Daramola et al.48 yielded ~ 30% improvement in tensile strength and compared with optimum enhancement of 72.9% in present study. The discrepancy may be on account of kenaf fiber distribution within matrix which aided stress distribution.Evidently, in mix 4 composites (4 wt% PSS), it is observed that KF loading demonstrated an uptrend in tensile strength from 5 to 20 wt%. Further addition of 30 wt% KF manifested slight depreciation in strength. By comparison, the tensile strength of mix 4 composites reinforced with 5 and 10 wt% KF yielded 8.5 and 3.9% lesser than their counterpart in mix 2 depicting the fact that the coalesce of 2 wt% PSS and 5 and 10 wt% KF amounts to strong cohesion and even stress distribution within the PP matrix as observed in Fig. The representative results for the statistical data showing the ANOVA results for hardness, impact strength, compressive strength, flexural strength, and Tensile strength are as presented in Tables A represents kenaf fiber input (KF), B represents particulate snail shell input (PSS).From Table Statistical analysis on impact strength is represented by ANOVA Table  and PareAs for compressive strength, contributions of particulate snail shell and interaction B*B, A*B had a significant effect on compressive strength Table . ParametParameters A, B, and A*B significantly influence tensile strength as depicted in Table  while AAThe various trend of the Taguchi analysis (main effect plot for S/N ratios) on the experimental results is presented in Fig. Figure Taguchi optimization was carried out simultaneously on hardness, impact strength, compressive, flexural, and tensile strengths using Minitab 19 application. It was applied to determine input parameters that will yield optimum performance for all properties. Figure the blend of 2 wt% PSS and 5, 10, 20, and 30 wt% KF ensued progressive enhancement of hardness, impact, compressive and flexural strength with 2 wt% PSS/30 wt% yielding 14.9, 46.4, 27.5, and 53.4% for each property respectively. For flexural strength, optimum improvement was attained when 2 wt% PSS was combined with 30 wt% KF. Meanwhile, tensile strength achieved an optimum improvement of 72.9% when 2 wt% PSS was combined with 20 wt% KF.presence of 4 wt.% PSS, 5, 10, 20, and 30 wt% KF engendered progressive enhancement of hardness, 4 wt% KF yielded 29.5 optimum improvements for impact strength while the same dosage yielded optimum enhancement of 36.9% over neat PP for compressive strength. Flexural strength was enhanced by admix of 5 and 10 wt% KF even as further addition of 20 and 30 wt% KF led to a depreciation in strength. Tensile strength showed progressive enhancement up to 20 wt% KF before a further decline.the blend of 10 wt% PSS and 5, 10, 20 and 30 wt% KF triggered enhancement of hardness, the same proportion spawned downward trend in impact, compressive, flexural, and tensile strength as proportion KF increased from 5 to 30%. Hence, intermix of 10 wt% PSS and kenaf fiber from 5 to 30 wt% are detrimental to properties of PP matrix.Taguchi optimization revealed an optimum mix proportion of 4 wt% PSS/10 wt% KF. Conclusively, particulate snail shell derived from African giant snail and kenaf bast fiber are good reinforcement in polypropylene in the development of a  cost effective but stronger biocomposites which can find applications in automobile and aerospace application.developed composite in this study showed better performance than their counterpart reinforced with hydroxylapatite and chitosan which are mostly used in reinforcement of biopolymers.Kenaf fiber (KF) and particulate snail shell (PSS) were added to polypropylene (PP) matrix with the view of improving the mechanical properties for engineering application, the following conclusions were arrived at;


Body mass index (BMI) is an inadequate marker of obesity, and cannot distinguish between fat mass, fat free mass and distribution of adipose tissue. The purpose of this study was twofold. First, to assess cross-sectional relationship of BMI with fat mass index (FMI), fat free mass index (FFMI) and ratio of fat mass to fat free mass (FM/FFM). Second, to study the association of FMI, FFMI and FM/FFM with physical function including sarcopenia, and cognition in pre-frail older adults.Cross-sectional study of 191 pre-frail participants ≥ 65 years, 57.1% females. Data was collected on demographics, cognition [Montreal Cognitive Assessment (MoCA)], function, frailty, calf circumference, handgrip strength (HGS), short physical performance battery (SPPB) and gait speed. Body composition was measured using InBody S10. FMI, FFMI and FM/FFM were classified into tertiles  with T1 classified as lowest and T3 highest tertile respectively and stratified by BMI.Higher FFMI and lower FM/FFM in the high BMI group were associated with better functional outcomes. Prevalence of low muscle mass was higher in the normal BMI group. FMI and FM/FFM were significantly higher in females and FFMI in males with significant gender differences except for FFMI in ≥ 80 years old. Small calf circumference was significantly less prevalent in the highest tertile of FMI, FM/FMI and FFMI. Prevalence of sarcopenic obesity and low physical function  were significantly higher in the highest FMI and FM/FFM tertile. Highest FFMI tertile group had higher physical function, higher MoCA scores, lower prevalence of sarcopenic obesity and sarcopenia, After adjustment, highest tertile of FFMI was associated with lower odds of sarcopenia especially in the high BMI group. Highest tertile of FM/FFM was associated with higher odds of sarcopenia. Higher BMI was associated with lower odds of sarcopenia.FFMI and FM/FFM may be a better predictor of functional outcomes in pre-frail older adults than BMI. Cut-off values for healthy BMI values and role of calf circumference as a screening tool for sarcopenia need to be validated in larger population. Health promotion intervention should focus on FFMI increment. Population ageing and rise in obesity prevalence worldwide are the two biggest risk factors for non-communicable diseases, including degenerative diseases, sarcopenia, frailty, dementia, increased morbidity, and mortality putting a strain on finite healthcare resources –5. PhysiAgeing is known to be associated with body composition changes such as increase in fat mass, decrease in fat free mass and skeletal muscle , 16, 17.Frailty is a multidimensional geriatric syndrome caused by a decline in physiological reserves that increases one’s vulnerability to stressors and associated with increased morbidity and mortality . FunctioThe impact and association of BMI and body composition especially fat mass, fat free mass and FM/FFM on physical function, sarcopenia and cognition in pre-frail older adults is still unclear. The purpose of this study was twofold. First, to assess cross-sectional relationship of BMI with fat mass index (FMI), fat free mass index (FFMI) and FM/FFM. Second, to study the association of FMI, FFMI and FM/FFM with physical function including sarcopenia, and cognition in pre-frail older adults.2 and prefrail were included in the analysis as numbers of those with BMI < 18.5 kg/m2 and/or frail were less than 10%.This was a cross-sectional sample consisted of 191 pre-frail older adults recruited from an ongoing study on effect of multidomain intervention at two primary care clinics i.e. Choa Chu Kang and Bukit Batok National University Polyclinic, Singapore (84.4%), Geriatric Clinic at National University Hospital and Alexandra Hospital, Singapore (5.2%) and community setting (10.4%). Participation was entirely voluntary with Choa Chu Kang National University Polyclinic being the control site. Inclusion criteria included participants who could provide consent and follow instructions as certified by primary care physician or study team, ambulant and pre-frail or frail. Exclusion criteria included nursing home residents and those who were either bedridden or wheelchair bound. Only those with BMI of ≥ 18.5 kg/mInterview questionnaire was administered by trained research staff on demographics, chronic diseases, medications, falls, function, cognition, frailty, sarcopenia, depression, and perceived health. Multimorbidity was defined as ≥ 2 or more self-reported chronic conditions. Three-minute nutritional screening (3-MinNS) tool was used to assess malnutrition risk . Katz acPhysical performance test included assessment of handgrip strength (HGS), gait speed and Short Physical Performance Battery test (SPPB). Maximum HGS was measured using Jamar hand dynamometer on the dominant arm in the seated position with elbow flexed at 90°. Poor HGS was defined according to the 2019 Asian Working Group for Sarcopenia criteria with cut-offs of 28kg for males and 18kg for female . The SPP2). Normal BMI cut-off was based on WHO recommendations for Asians  and whole-body phase angle. Inbody bioelectrical impedance analyzer has been validated in different populations including older adults and Asians  by height squared , with an addition of BMI for logistic regression. Adjustments were made for age, gender and physical activity for logistic regression, and age and physical activity for multiple linear regression. Odds ratios and B-coefficients were obtained as test statistics for logistic and multiple linear regression respectively, with 95% confidence intervals for all models. One-Way ANOVA with Tukey test and Welch ANOVA with Games-Howell were also used in The studies involving human participants were reviewed and approved by The National Healthcare Group Domain Specific Review Board to be conducted at the Choa Chu Kang and Bukit Batok National University Polyclinic, Geriatric Clinic at National University Hospital and Alexandra Hospital and community setting in Singapore. The patients/participants provided their written informed consent to participate in this study.2 for FMI, 28.3 ± 4.4kg/m2 for FFMI and 29.7 ± 3.6kg/m2 for FM/FMI respectively. The prevalence of small calf circumference was significantly higher in the lowest tertile compared with highest tertile for FMI (37.9% vs 4.9%), FFMI (40.0% vs 6.3%) and FM/FFM (29.0% vs 3.6%). Diabetes prevalence was significantly higher in all the highest tertile compared with lowest tertile for FMI, FFMI and FM/FFM, 63.6% vs 42.4%, 62.1% vs 33.9% and 65.6% vs 46.9% respectively. There was significantly higher prevalence of at least 1 ADL impairment in the higher FMI tertiles, 22.7% in FMI T3, 19.7% in FMI T2 compared with 6.8% in FMI T1. Physical activity levels were significantly higher in the lowest FMI and FM/FFM tertile and highest FFMI tertile.The sample comprised of 82 men and 109 women aged ≥ 65 years with a mean age of 73.0 ± 5.7 years and 57.1% were females. They were all classified as pre-frail based on the FRAIL scale. Demographics, clinical characteristics, functional measures, cognitive scores, perceived health, and body composition of the study population stratified according to FMI, FFMI and FM/FMI tertiles are shown in vs 16.9%) and FM/FFM (55.7% vs 15.6%). On the contrary, 45.8% of the lowest FFMI tertile compared with 13.6% of the highest tertile were classified as sarcopenic obesity. SARC-F scores were 3-fold higher in the highest tertile compared with lowest tertile for FMI (2.3 ± 2.0 vs 0.8 ± 1.4) and FM/FFM (2.4 ± 2.0 vs. 0.8 ± 1.3).Amongst the lowest FFMI tertile group, 55.2% were classified as sarcopenic compared with 4.5% in the highest tertile group. There were no significant differences between tertiles in the prevalence of sarcopenia for FMI and FM/FFM. Prevalence of sarcopenic obesity was highest in the highest tertile compared with lowest tertile for FMI (51.5% vs 19.2kg ± 5.8) and FM/FMI (26.2kg ± 6.7 vs 18.6kg ± 5.7). For FFMI, HGS was significantly higher in the highest tertile compared with lowest tertile, 26.4kg ± 7.3 vs 18.4kg ± 5.1 respectively. While there was no significant difference in mean gait speed amongst FFMI tertiles, it was significantly lower for FMI T2 and T3 compared with T1  and FM/FFM T2 and T3 compared with T1 . SPPB results were significantly higher in the FMI T1 (10.3 ± 1.8) compared with T2 (9.3 ± 2.6) and T3 (9.3 ± 2.3). For FM/FFM, SPPB scores were also significantly higher in the T1(10.3 ± 1.8) compared with T2 (9.3 ± 2.6) and T3 (9.2 ± 2.4). For FFMI, significant difference in SPPB scores were only observed between T1 (9.2 ± 2.4) and T3 (10.3 ± 2.0). Mean MoCA scores were significantly higher in the highest FFMI tertile compared with lowest, 25.8 ± 3.2 and 23.6 ± 5.3 respectively.HGS was significantly higher in the lowest tertile compared with highest tertile for FMI (24.9kg ± 6.7 2) compared to T1 (5.9 ± 1.1kg/m2) and T2 (7.1 ± 1.3kg/m2). The reverse was true for FM/FFM, where ASM was significantly higher in T1 (7.4 ± 1.7 kg/m2) compared with T3 (6.7 ± 1.2kg/m2). There was significantly higher prevalence of low muscle mass in the lowest FFMI tertile (67.8%) compared with highest FFMI tertile (4.5%). Whole body phase angle was significantly higher in the lowest compared with the highest FMI tertile (5.2 ± 0.9 vs 4.8 ± 0.8) and FM/FMI tertile  was used as reference Table 3.The models were adjusted for age and physical activity Table 4.Our study has shown that body composition especially higher FFMI and lower FM/FFM were associated with better functional outcomes in the pre-frail. The differences in outcomes were more significant in the higher BMI group. Those with high BMI and in the highest FFMI tertile had much lower odds of being classified as sarcopenic. On the contrary, amongst those with high BMI, highest FM/FFM tertile was associated with higher odds of sarcopenia. There was a higher prevalence of low muscle mass in our participants with normal BMI. Small calf circumference was significantly less prevalent in the highest FMI, FM/FMI and FFMI tertile. Sarcopenic obesity was significantly more prevalent in the highest FMI and FM/FMI tertile. Higher fat mass was associated with slower gait speed.Until recently, BMI has been reported as a useful predictor of metabolic diseases but studies have only shown moderate correlation for diabetes and other chronic diseases . BMI doeThere were gender differences where FFMI was significantly higher and FM/FFM significantly lower in male while body fat percentage and FMI being significantly higher in females across all age groups. Similar findings have been shown even in normal population . Unlike Chronic low-grade inflammation with ageing and obesity is associated with diabetes and frailty . Almost For those in highest the FMI, FM/FFM and lowest FFMI tertile, only 1 in 10 fulfilled the WHO physical activity recommendations. Sedentary lifestyle has been associated with increase in fat mass and weight gain . Weight Increasingly, phase angle is being considered as an indicator of nutritional status, muscle quality, muscle size and function, cellular integrity, and predictor of mortality –67. PhasCalf circumference is positively correlated with muscle mass, associated with nutritional status and disability, and recommended by the Asian Working Group for Sarcopenia as a screening tool for sarcopenia , 69, 70.There is still an ongoing debate on the diagnosis and the definition of sarcopenia which continues to evolve. Body composition including fat mass and fat free mass plays an important role in diagnosis and management of sarcopenia. Most sarcopenia definitions have included gender specific cut-offs for ASM and applied the same cut-off for sarcopenia across the BMI range from normal to obese without considering the relative contribution of FMI and/or FFMI to physiological function and adipose tissue dysfunction. Muscle quality was only recently added to the definition in 2019 . Loss ofThe strength of our study includes involvement of community dwelling older adults and objective measurements of muscle strength, and body composition. The limitation is the cross-sectional design which did not allow us to evaluate longitudinal changes of physical function with change in FMI, FFMI and FM/FFM. Our study included only pre-frail participants with normal or high BMI and findings cannot be generalized to the entire population. In addition, we did not adjust for nutrition intake. The subgroup sample size is small as this was not the primary aim of the multidomain study. Nonetheless, high FFMI did correlate with better physical and cognitive function in this group. Low muscle mass was more prevalent in the normal BMI group in the pre-frail. We did not investigate the multifactorial causes of sarcopenia. Lastly, body composition measured by BIA may be affected by hydration, edema and fasting.While higher FFMI and lower FMI/FMI was beneficial and associated with better physical and cognitive function, there is an urgent need to have normative data based on age, ethnicity, gender, and validation carried out for risk prediction of cardiometabolic, sarcopenia and musculo-skeletal disorders .BMI cannot distinguish between fat mass, fat free mass and fat distribution. FFMI and FM/FFM may be a better measure of cognitive and functional outcome in older adults. In pre-frail older adults, there was a higher prevalence of low muscle mass in the normal BMI group. Higher BMI was associated with lower odds of sarcopenia in the pre-frail. Higher phase angle and body cell mass was more prevalent in the lower FM, FM/FFM and higher FFMI tertile which are indicators of nutritional status, muscle quality, cellular integrity and mortality. The role of calf circumference as a screening tool for sarcopenia in those with high BMI and cut-off values for healthy BMI values needs to be validated in larger population. Health promotion and frailty prevention intervention should focus on FFMI increment.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Domain Specific Review Board, National Healthcare Group, Singapore. The patients/participants provided their written informed consent to participate in this study.Data cleaning and analysis: RM and MW. Figure and table design: RM and MW. Writing and methodology: RM. Writing, review and editing: RM, MW, MC, LT, BW, RH, JS, LA, SS, and JM. All authors contributed to the article and approved the submitted version.This study is sub-study of a larger study that has been funded by Ministry of Health of Singapore: Healthy Ageing Innovation Grant under National Innovation Challenge on Active and Confident Ageing (Award No: MOH/NIC/HAIG02/2017) and National Medical Research Council (HSRG-HP17Jun003).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


DCISionRT identified women with elevated recurrence risk who benefited substantially from RT after BCS. In addition, the test identified women with low recurrence risk and little benefit from RT. These results support our conclusions that knowledge of the individual risk and benefit from RT provided by the test can help clinicians and patients make individualized treatment decisions for women diagnosed with DCIS.Despite clinical advancements in the diagnosis and treatment of DCIS, tailoring individual treatment for women diagnosed with DCIS remains an unmet clinical need. Definitive predictive tools that can predict who will or not benefit from radiation therapy (RT) after breast conserving surgery (BCS) remains elusive. Here, we used a prospective–retrospective design to validate DCISionRT®, in the SweDCIS randomized trial. Women were randomly assigned to RT or not after BCS, between 1987 and 2000. Tumor blocks were collected, and slides were sent to PreludeDxTM for testing. In 504 women with complete data and negative margins, DCISionRT divided 52% women into Elevated (DS > 3) and 48% in Low (DS ≤ 3) Risk groups. In the Elevated Risk group, RT significantly decreased relative 10-year ipsilateral total recurrence (TotBE) and 10-year ipsilateral invasive recurrence (InvBE) rates, HR 0.32 and HR 0.24, with absolute decreases of 15.5% and 9.3%. In the Low Risk group, there were no significant risk differences observed with radiotherapy. Using a cutoff of DS > 3.0, the test was not predictive for RT benefit (p = 0.093); however, above DS > 2.8 RT benefit was greater for InvBE (interaction p = 0.038). Recurrences at 10 years without radiotherapy increased significantly per 5 DS units (TotBE HR:1.5 and InvBE HR:1.5). Continuous DS was prognostic for TotBE risk although categorical DS did not reach significance. Absolute 10-year TotBE and InvBE risks appear sufficiently different to indicate that DCISionRT can aid physicians in selecting individualized adjuvant DCIS treatment strategies. Further analyses are planned in combined cohorts to increase statistical power.Prediction of radiotherapy (RT) benefit after breast-conserving surgery (BCS) for DCIS is crucial. The aim was to validate a biosignature, DCISionRT Four randomized clinical trials (RCT) demonstrated that 30% of patients diagnosed with breast ductal carcinoma in situ (DCIS) recur after breast conserving surgery (BCS) at 10 years ,3,4,5. S® biosignature  combines biomarkers with clinicopathological factors and reports a continuous Decision Score (DS) on a scale (0–10) with corresponding 10-year risks after BCS with and without RT [The DCISionRTthout RT ,8. This thout RT  where itHere we validated DCISionRT for prognostic risk assessment and for RT benefit in the SweDCIS RCT with long-term follow up ,10.n = 247 and 257, 49% and 51%), except that slightly more women over 70 years of age received RT in the validation cohort (st quartile to 3rd quartile: 15.2 to 20.5 years), which was longer than the 10 years used to assess outcomes in this study. After 10 years, there were 59 DCIS and 31 invasive events in the validation cohort.  for TotBE and a HR of 1.5 per 5 DS units (95% CI 0.7 to 2.8) for InvBE. A categorical DS Low Risk group (DS ≤ 3) was pre-specified to identify patients with 10-year 10% TotBE risk and 6% InvBE risk [n = 240/504, 48%), and the remaining into the Elevated Risk group . The percentage of women with Low and Elevated Risk within different clinicopathologic factors is reported in DCISionRT classified approximately half of the women into the Low Risk group . For InvBE, the relative risk increased, though not statistically significantly, with a HR of 1.5 per 5 DS units (95% CI 0.7–2.8). For categorical DS Risk groups (DS ≤ 3 vs DS > 3), the HRs for 10-year event rates were 1.5 (95% CI 0.9–2.6) for TotBE and 1.7 (95% CI 0.7–4.2) for InvBE. In the Elevated Risk group, there was a statistically significant relative RT benefit with a 10-year TotBE HR of 0.32 (95% CI 0.17 to 0.58) and InvBE HR of 0.24 (95% CI 0.08 to 0.74), see p > 0.05) depending on DS threshold for TotBE rate but did vary significantly (p < 0.05) depending on DS threshold for InvBE rate. There was a statistically significant interaction (p = 0.035) for a DS threshold of 2.8 for 10-year InvBE rate. The RT benefit for 10-year InvBE rate was statistically significant above a DS threshold of 2.8 with HR 0.22 (0.07 to 0.68) but was not statistically significant below a DS threshold of 2.8 with HR 1.15 (0.38–3.41).According to the predefined statistical analysis plan (SAP) we assessed an optimal threshold for predicting differential relative RT effect by DS. The multiplicative interaction of DS with RT (RT: DS > x) was assessed for DS thresholds between 1.0 and 3.0, One key finding from this validation of DCISionRT was that continuous DS was prognostic for 10-year recurrence rates. Also, using the pre-specified DS threshold of 3.0 to define DS Low and Elevated Risk groups, approximately 50% of women were classified into the Elevated Risk group. In the DS Elevated Risk group, the addition of RT reduced absolute 10-year rates for TotBE by 15.9%, and for InvBE by 9.4%. This represented a relative RT benefit of approximately 70% for both DCIS and invasive recurrences, consistent with prior studies . This obBesides being performed in a randomized cohort, with a pre-specified SAP, this study has some additional strengths. Only 3% of the women in this validation received tamoxifen, which makes the influence of endocrine therapy negligible . TamoxifOur results are consistent with prior validations of DCISionRT, including the independent validation by Kaiser Permanente and the cross-validation in cohorts from Uppsala University and the University of Massachusetts ,8. In paClinicopathological factors have been explored to identify low-risk patients with limited benefit from RT. However, in randomized trials, including SweDCIS, the relative risk reduction by RT has been about 50% or more in all risk groups ,13,14. IWhile depending on local practices, RT may not be recommended for women with “low-risk” DCIS. However, 30–40% of these women with “low-risk” clinicopathology were re-classified into the DS Elevated Risk group. DCISionRT’s biologic approach and non-linear algorithm may be an explanation why the test is not only prognostic, but also seems to provide predictive value. DCISionRT may aid physicians and patients to make individualized treatment decisions, as exemplified by a 42% change in RT recommendations in an ongoing prospective registry study .A limitation of the present validation is that SweDCIS may not be representative of modern practice. Women diagnosed after 1995 are expected to have lower risks profiles, which was adjusted for in multivariable analysis. Whether or not women were included may have depended on clinicopathological factors or changes in mammographic screening over time. However, 79% of all DCIS cases in SweDCIS were detected by screening, in line with contemporary data , and theAnother limitation was that fewer patients than in the entire SweDCIS RCT were available for analysis with a corresponding lower number of events, resulting in wider confidence intervals and preventing further subset analyses. However, an analysis including multiple cohorts is planned, to assess test performance within different clinicopathological risk groups, such as data presented at SABCS 2020 within patients meeting RTOG 9804 criteria .The DCISionRT biosignature combines protein expression  by immunohistochemistry with clinicopathologic factors . The continuous DS is calculated using a proprietary non-linear algorithm to account for interactions between factors in order to better interpret complex biological information. The biological signature was parameterized and tested using multiple cross-validations and produced a consensus continuous risk score on a scale from zero to ten, termed Decision Score (DS). A risk threshold was selected using the training datasets in the cross-validated development with the goal of identifying an average 10-year IBE risk of 10% and an IBC risk of 6% or less. Patients with a score greater than the threshold belonged to the Elevated Risk Group. The threshold between the Low and Elevated Groups was scaled to 3, with the Low Risk Group including patients with DS ≤ 3, and the Elevated Risk Group including patients with DS > 3 .Test performance was evaluated on subjects from the SweDCIS RCT ,10,11 usThe objectives were to assess the association of continuous DS with 10-year recurrence rates in women treated without RT, and to assess the association of RT and 10-year recurrence rates within categorical DS Risk groups, which were prespecified using a DS threshold of 3.0 to identify a low risk group with 10-year risks of 10% TotBE and 6% InvBE . Other pArchived formalin-fixed paraffin-embedded tissue blocks were collected from 26 Swedish pathology departments. H&E slides were used to identify representative blocks. Tissue samples were processed in Sweden, and 4-µm whole sections on slides were sent to PreludeDx. DCISionRT testing was performed by PreludeDx while blinded to patient outcomes. Biomarker expression, nuclear grade (NG), and tumor size were determined after verification of DCIS without invasive disease by board-certified pathologists. PreludeDx transmitted biosignature data and test results that were combined with clinical and outcome data in Sweden. Size was defined according to the following priority based on SweDCIS case report forms: microscopic size followed by macroscopic, mammographic, and size from new sections. Surgical margin was reported as negative, positive, or unknown. Outcomes and clinicopathology were determined using the SweDCIS database. Follow-up was completed prior to 15 December 2012. Our database was created for test validation following REMARK criteria. The original SweDCIS RCT included 1046 Swedish women diagnosed from 1987 to 2000 who were randomly assigned to whole breast RT or no RT after BCS . Tumor bAnalyses were conducted for 10-year TotBE and InvBE rates. Hazard ratios (HR) for association of RT, year of diagnosis and clinicopathologic factors with 10-year rates were determined by multivariable Cox proportional hazards (CPH) analysis. In the validation cohort, HRs for the association of RT with 10-year rates were determined by CPH within categorical DS Risk groups . HRs for the association of continuous DS with 10-year rates were determined by CPH for women treated without RT. Absolute 10-year rates were calculated by Kaplan–Meier analyses, and rates and differences with 95% confidence intervals (CI) were calculated using Cox proportional models by means of flexible parametric survival model (stpm2)  and stanClinicopathologic factors were summarized by counts and percentages for patient subsets, and classification of patients by DS Risk group within individual clinicopathologic risk factors or RTOG 9804 criteria ,15 was sRadiotherapy benefit was statistically significant in patients with elevated DS but not in patients with low DS by DCISionRT. Continuous DS was prognostic for TotBE risk although categorical DS did not reach significance. Absolute 10-year TotBE and InvBE risks appeared sufficiently different to indicate that DCISionRT can aid physicians in selecting individualized adjuvant DCIS treatment strategies. Further analyses are planned in combined cohorts to increase statistical power.


The aim of this study was to report 36-month longitudinal changes using the North Star Ambulatory Assessment (NSAA) in ambulant patients affected by Duchenne muscular dystrophy amenable to skip exons 44, 45, 51 or 53.We included 101 patients, 34 had deletions amenable to skip exon 44, 25 exon 45, 19 exon 51, and 28 exon 53, not recruited in any ongoing clinical trials. Five patients were counted to skip exon 51 and 53 since they had a single deletion of exon 52.p≤0.001).The difference between subgroups  was significant at 12 (p = 0.043), 24 (p = 0.005) and 36 months (Mutations amenable to skip exons 53 and 51 had lower baseline values and more negative changes than the other subgroups while those amenable to skip exon 44 had higher scores both at baseline and at follow up.Our results confirm different progression of disease in subgroups of patients with deletions amenable to skip different exons. This information is relevant as current long term clinical trials are using the NSAA in these subgroups of mutations. The North Star Ambulatory Assessment (NSAA) is increasingly used as a primary or secondary outcome measure in clinical trials in Duchenne muscular dystrophy (DMD), as it combines a reliable assessment of several aspects of motor function with a structured validated format , 2. SeveThe aim of our collaborative effort was to obtain longitudinal prospective changes over 36 months in the NSAA in a large cohort of DMD patients amenable to skip exons 44, 45, 51 and 53. We also aimed to establish if within each genetic subgroup, age, Time to Raise from Floor (TRF) or baseline NSAA could help to identify patients at risk of loss of ambulation or reduced ambulatory activity.The study is part of a longitudinal multi-centric cohort effort involving 12 tertiary neuromuscular centers in Italy, two centers in the United Kingdom, one in France, one in the Netherlands and one in Belgium. Patients were recruited between January 2008 and September 2017 and followed for at least three years. Inclusion criteria at baseline were: genetically confirmed DMD diagnosis, patient still ambulant and able to walk independently for at least 150 meters at baseline, no severe or moderate intellectual disability or behavioral problems.In this manuscript, only patients with deletions amenable to skip exons 44, 45, 51 and 53 and who are not taking part in interventional trials/receive commercially available drugs were included. Each center included in the project had approval from the local Ethical Committee .The informed consent signed by parents of participants and patients included in this study declared that all data relevant to the disease  and collected as part of clinical routine could be included, in an anonymous format, in observational studies defining natural history.The NSAA is an ordinal scale consisting of 17 items, ranging from standing to running. It includes several items assessing abilities such as head raise and standing on heels and a number of dynamic activities such as hopping or running . Each itAs part of the clinical routine, patients were seen at least every 12 months. Data were collected from recruitment (baseline) and at 12, 24, 36-months after. Details on functional assessment training and of the inter-observer reliability among physiotherapists have already been reported [As part of this study, all boys were asked to perform the timed items, including time to rise from the floor (TRF). The TRF was performed recording the time (seconds) taken to complete the task of rising from supine to full standing .The boys who were able to perform the task were subdivided into two groups, based on the ability to perform the task in less or more than 6 seconds as used in previous studies . Boys thThe NSAA was evaluated longitudinally at 12, 24 and 36 months after baseline. Descriptive statistics  were used. Descriptive analysis also included assessment of patients who lost ambulation  or who reached a NSAA score <6, associated with limited functional ability.U test was used to compare differences between groups on the NSAA at baseline and at 12, 24, 36-month assessments or their changes from baseline. The test was performed according to age subgroups , TRF at baseline  or NSAA level at baseline . Chi-square test was used to analyze TRF  distribution among the skipping subgroup .Mann–Whitney Analysis of variance model (ANOVA) with Bonferroni correction was used to assess heterogeneity among groups of the NSAA at baseline and at 12, 24, 36-month assessments or their changes from baseline. The test was performed according to the type of skipping exons 44, 45, 51, 53 and the steroids regimen at baseline . For all the analysis the p-value was set at < 0.05.Of the 131 DMD patients who had deletions amenable to skip exons 44, 45, 51 and 53, 101  fulfilled the inclusion criteria and entered the study. The remaining 30 were not recruited either because of participation in clinical trials (n = 22), because they had been followed for less than three years (n = 4), or were lost at follow up (n = 4).Thirty-four of the 101 had deletions amenable to skip for exon 44, 25 amenable to skip for exon 45, 19 had deletions amenable to skip for 51, and 28 had deletions amenable to skip exon 53. Of the patients with a single deletion of exon 52, five were counted in both subgroups amenable to skip exon 51 and 53. Of the 101 boys, 43 were under 7 years of age at the time of the first assessment , 58 were above or equal to 7 years . None of the patients discontinued steroid treatment during the study.A difference on NSAA scores was found between the groups under and above 7 years of age at 12 months (p = 0.001), at 24 months (p<0.001) and at 36 months (p<0.001), with patients ≥7 years of age at baseline having lower NSAA mean scores; no statistical difference was found comparing baseline values .A difference on NSAA scores was also found between the groups under and above a baseline score of 22 on the NSAA, at 12 months (p<0.001), at 24 months (p<0.001) and at 36 months (p< 0.001), with patients performing a score ≤22 having lower NSAA mean scores.Of the 101 patients, 21 lost ambulation within 36 months from baseline. The mean age at loss of ambulation was 11.4 years (SD±1.9).In the group of patients < 7 years of age at baseline, a difference on the NSAA scores was found among the different steroids’ groups  at baseline (p = 0.040) and at 36 months assessments (p = 0.014) but not at 12- or 24-months assessments. Post hoc comparisons using the Bonferroni correction indicated that, at baseline, the mean score for the steroid’s naïve patients  was significantly lower than the patients doing daily steroid regime  (p = 0.039). At 36 months, the mean scores for the alternate steroid regime  was significantly lower than the mean scores for the daily steroid regime  (p = 0.025).In the group of patients ≥ 7 years of age at baseline, no difference on the NSAA was found among the different steroids’ regimen groups  at baseline, 12 or 24 months or at 36 months assessments.Sixty-six of the 101 patients had TRF below 6 sec and 32 had TRF above or equal to 6 seconds at baseline. No data were available from 3 patients .p≤0.001), 12 months (p≤0.001), 24 months (p≤0.001) and at 36 months (p≤0.001), with patients above 6 seconds having lower mean scores and higher mean changes on the NSAA  and 24 of the 32 (75%) had scores ≤22. In the group with baseline TRF <6 sec, the NSAA scores at baseline ranged between 13 and 34  and 9 of the 66 (13%) had scores ≤22.The mean NSAA was 25.9 at baseline. Mean changes from baseline were +0.1 at 12 month, -1.8 mt at 24 months and– 3.3 at 36 months. Sixteen were younger than 7 years of age and 18 were above or equal to 7 years . TRF at baseline was ≥6 sec in one and unknown in the other. TRF at baseline ≥6 sec was also present in 3/32 (9%) who did not lose ambulation.p = 0.005), 12 months (p≤0.001), 24 months (p≤0.001) and 36 months (p≤0.001), with patients above 6 seconds having lower mean scores in the NSAA assessment.A difference in the NSAA scores was found when subdividing the groups into below and above 6 seconds TRF at baseline (p≤0.001), 24-month (p≤0.002), and 36-month (p≤0.001) changes, with patients above 6 seconds having higher mean changes scores.A difference was also found in the NSAA changes at 12-month  at baseline, 12, 24, 36 months or their changes, while in the group of patients <7 years of age at baseline, a trend was observed at 12-month changes (p = 0.036) and 36-month changes (p = 0.037), but this result was not significant after Bonferroni correction.The mean NSAA was 24.1 at baseline. Mean changes from baseline were -1.3 at 12 months, -2.9 at 24 months and -2.3 mt at 36 months. Twelve were younger than 7 years of age and 13 were above or equal to 7 years . All three (100%) had a TRF ≥6 sec at baseline. TRF at baseline ≥6 sec was also present in 4/22 (18%) who did not lose ambulation.p = 0.021), 12 months (p = 0.002), 24 months (p = 0.007), at 36 months (p = 0.001), with patients above 6 seconds having lower mean scores in the NSAA assessment.A difference in the NSAA scores was found when subdividing the groups into below and above 6 seconds TRF at baseline (p = 0.017) but not at 24-month (p = 0.64) nor at 36-month follow up (p = 0.64), with patients above 6 seconds having higher mean changes scores. No difference in the NSAA was found among the different steroids’ regimen groups  at baseline, 12, 24, 36 months or their changes, irrespective of age at baseline (<7 or ≥7 years).A difference was also found in the NSAA changes at 12-month . Six had a TRF at baseline ≥6 sec at baseline and in the remaining one TRF at baseline was unknown. TRF at baseline ≥6 sec was also present in 2/13 (15%) who did not lose ambulation.p = 0.001), 12 months (p = 0.001), 24 months (p = 0.002), at 36 months (p = 0.001), with patients above 6 seconds having lower mean scores on the NSAA assessment.A difference on the NSAA scores was found when subdividing the groups into below and above 6 seconds TRF at baseline (p>0.05). No difference on the NSAA was found among the different steroids’ groups  at baseline, 12, 24, 36 months or their changes, irrespective of age at baseline (<7 or ≥7 years).A difference on the NSAA scores was not found when subdividing the groups into below and above 6 seconds TRF at 12, 24 or 36-month changes . TRF at baseline ≥6 sec was also present in 8/19 (42%) who did not lose ambulation.p≤0.001), 12 months (p≤0.001), 24 months (p≤0.001) at 36 months (p = 0.001), with patients above 6 seconds having the lowest mean scores to the NSAA assessment.A difference on the NSAA scores was found when subdividing the groups into below and above 6 seconds TRF at baseline (p>0.05). No difference on the NSAA was found among the different steroids’ groups  at baseline, 12, 24, 36 months or their changes, irrespective of age at baseline (<7 or ≥7 years).A difference on the NSAA scores was not found when subdividing the groups into below and above 6 seconds TRF at 12, 24 or 36-month changes (2(3 = 14.55) p = 0.002), with 12% and 38% of patients amenable to skip 44 and 45 respectively having a TRF ≥6 seconds at baseline compared to 44% and 57% of patients amenable to skip 51 and 53 respectively. TRF ≥6 seconds was reached at a mean age of 9.2 years (SD ±1.5) in patients amenable to skip exon 44, at 8.7 years (SD ±2.7) in patients amenable to skip exon 45, at 8.6 years (SD ±2.3) in patients amenable to skip exon 51 and at 8.0 years (SD ±2.1) in patients amenable to skip exon 53. The difference among individual subgroups was only significant between those amenable to skip exon 44 and 53.The proportion of subjects who had a TRF <6 or ≥6 at baseline differ by skip group (Xp≤0.001). Post hoc comparisons using the Bonferroni correction indicated that at 12 months there was no significant difference among individual subgroups. At 24 months, there was a difference only between patients amenable to skip exon 44  and patients amenable to skip exon 53  (p = 0.016). At 36 months, there was a difference between patients amenable to skip exon 44  and both patients amenable to skip exon 51  (p = 0.001) and exon 53  (p = 0.001). There was also a difference between patients amenable to skip exon 45  and those amenable to skip exon 51 (p = 0.001) or exon 53  (p<0.001). There was no difference between patients amenable to skip exon 44  and 45  or between patients amenable to skip exon 51  and 53 .When we analyzed the NSAA mean changes from baseline, there was a significant difference between deletions amenable to skip exons 44, 45, 51 or 53 at 12 (p = 0.043), 24 (p = 0.005) and 36 months  than in those amenable to skip exon 51 and 53 (37% and 32%). More generally, NSAA scores lower than 6, associated with very restricted functional ability were found in many patients amenable to skip exon 53 and 51 (32% and 42% respectively), and were often observed already soon after the age of 8–9 years while such low scores occurred only in two patients amenable to skip exon 44 after the age of 12 years and in three patients amenable to skip exon 45 between the age of 9 and 12 years.These results are consistent with other studies investigating differences between patients eligible to skip different exons. In particular, patients eligible to skip exon 44 are reported to have higher level of revertant fibers and traces of dystrophin expression than those eligible to skip exon 51 or 53 . This haThe difference among subgroups did not appear to be related to steroid regime but the interpretation of these results is limited by the fact that when identifying different regimes in the small subgroups, the numbers were very small.In this paper we were also interested to establish clinical features that may help to predict functional decline in the different subgroups. In agreement with previous findings reporting that TRF can be used to predict functional decline and loss of ambulation , 18, we This occurred in all genetic subgroups but the age when patients reached the point of TRF decline was different, as patients amenable to skip 51 and 53 reached TRF>6 sec more often and at an earlier age compared to those amenable to skip 45 and 44. Interestingly, most patients (83%) with TRF ≥6 sec also had NSAA scores ≤22 that had previously been reported to be associated with increased risk of loss of ambulation within 2 years.Our study, using the NSAA, confirmed that subgroups of DMD patients amenable to skip different exons have a different progression over 3 years, as previously reported in a study assessing the 6MWT changes.Unfortunately, the cohort in the present study was not completely overlapping with the cohort reported in the 3-year 6MWT study , as in s


S. aureus enterotoxins. Nowadays, many researchers worldwide noted an emergence of resistant strains such as Staphylococci particularly for the antibiotic methicillin. Therefore, this study was aimed to determine the existence of Staphylococcus aureus and its enterotoxins, mecA genes, in selected food samples. A total of 400 selected food samples were collected from different areas in Khartoum State. The selected foods included cheese, meat products, fish, and raw milk. One hundred samples from each type of food were cultivated, and the resultant growth yielded 137 (34.25%) S. aureus, 126 (31.5%) bacteria other than S. aureus, and 137 (34.25%) yielded no growth. Eighty-four of the 137 S. aureus isolates were randomly selected and tested for the presence of mecA and enterotoxin genes. The oxacillin sensitivity test showed that 15 (11%) of 137 S. aureus isolates were oxacillin resistant. The PCR assay showed that the mecA gene was detected in 15 of 84 (17%) S. aureus isolates. Simultaneously, only 2 (2.385%) out of 84 S. aureus isolates showed an enterotoxin B gene product. There was a relatively moderate prevalence of methicillin-resistant Staphylococcus aureus with very low frequency of enterotoxin B gene in different kinds of selected food samples collected from Khartoum State. These findings elucidate the increased risk on public in Khartoum being affected by Staphylococcal food poisoning upon consumption of dairy or meat products prepared in unhygienic conditions that could lead to intoxication by Staphylococcus aureus enterotoxins.Staphylococcal food poisoning is an intoxication that results from the consumption of improperly prepared or stored foods containing sufficient amounts of one or more preformed  Foodborne diseases (FBD) remain as one of the greatest concerns in public health and food safety; they are caused by many pathogens that contaminate food and food products . Many foStaphylococcus aureus and are ideal for enterotoxin production including milk, meat, meat products, dairy products, and ready-to-eat food [Staphylococcal food poisoning (SFP) is an intoxication that results from the consumption of improperly prepared or stored foods containing sufficient amounts of one or more preformed enterotoxins , 4. A wieat food , 6.Staphylococcus aureus may produce a large variety of enterotoxins, 95% of food poisoning outbreaks are caused by classical enterotoxins A, B, C, D, and E [Staphylococcus aureus have the ability to resist heat and drying; hence, it can easily contaminate foods. This contamination might come from food handlers or from the environment where the bacteria multiply and release toxins in uncooked or inadequately cooked foods, especially if the foods are unrefrigerated [Although D, and E . Since tD, and E . Some stigerated .Nonhygienic handling practices, working conditions, and improper storage and refrigeration can all increase the opportunity for food contamination. So, it is important to follow the standard practices in food handling such as hand washing, proper cooking, proper storage, and others to reduce or prevent food contamination , 12.Staphylococcus aureus in food poisoning and the presence of enterotoxins in mecA genes in common consumed foods in Khartoum State. Hence, this study was conducted to determine the prevalence of enterotoxins and mecA genes in foods commonly consumed in Khartoum State, Sudan.There is paucity of information in Sudan regarding the role A total of 400 samples were collected from different areas in Khartoum State , during 2018. The type of foods included cheese, meat products, fish, and raw milk. Each sample was aseptically collected, fifteen grams of cheese samples were collected from different retailers by using sterile container, meat samples were collected randomly from supermarkets and restaurants using disposable blades, a small piece of raw meat had been splitted and transferred to the lab in sterile containers, small pieces of fish inner tissues were collected by a sterile blade and placed in sterile plain containers, and milk samples were collected in sterile containers and stored in a refrigerator at 4°C in the microbiology laboratory until examined.Staphylococcus aureus was confirmed based on colony morphology, Gram's reaction, and other biochemical tests including the catalase test, coagulase test, and DNase test.Meat, fish, and cheese samples were enriched in peptone water. The raw milk and the enriched peptone water samples were swabbed and inoculated in blood agar medium, mannitol salt agar medium, and MacConkey's agar medium and incubated aerobically at 37°C for 24–48 hrs. The presence of μg), gentamicin (10 μg), oxacillin (5 μg), and vancomycin (30 μg). The S. aureus ATCC 52923 control strain was used.The antimicrobial susceptibility test was performed by the disk diffusion method using Mueller–Hinton agar plates (oxoid) according to , where 4μl of sterile distilled water, and 10 μl of proteinase K was added and incubated at 6°C for 60 minutes. Then, it was incubated at 100°C in a waterbath for 15 minutes, and the suspension was centrifuged at high speed (10000 rpm for 10 min). The supernatant containing the genomic DNA was transferred into a fresh sterile Eppendorf tube and stored at −20°C until to be used for PCR [DNA was extracted by the simple boiling method, in which the extracted product was obtained from overnight isolates on nutrient agar. A loop full of bacterial colony was picked from an isolate and suspended in 300  for PCR .S. aureus by using specific housekeeping gene primer (16s) as shown in μL of a mixture containing 2 μL Maxime PCR Premix, 0.5 μL of each primer, 2 μL of template DNA, and 20 μL of double distilled water. The amplification conditions included three steps: initial denaturation at 94°C for 5 min; 35 successful cycles of denaturation at 94°C for 45 sec, annealing at 50°C for 45 sec, and extension at 72°C for 45 sec; and the final extension at 72°C for 7 min [All samples were confirmed as or 7 min .μL of mixture containing 2 μL Maxime PCR PreMix, 0.5 μL of each of the toxic gene-specific primers (5 μL), 2 μL of template DNA, and 16 μL of double distilled water. The amplification conditions included three steps: initial denaturation at 94°C for 5 min; 35 successful cycles of denaturation at 94°C for 45 sec, annealing at 50°C for 45 sec, and extension at 72°C for 45 sec; and the final extension at 72°C for 7 min [Multiplex PCR amplification was performed using the CLASSIC K960 (UK) thermocycler. PCR amplification of Staphylococcal enterotoxins (SE) genes, namely, sea, seb, sec, sed, and see, was performed using the Maxime PCR PreMix kit  and specific primers listed in or 7 min .μL of mixture containing 2 μL Maxime PCR PreMix, 0.5 μL of each of the gene-specific primers (5 μL), 2 μL of template DNA, and 13 μL of double distilled water. The amplification conditions included three steps: initial denaturation at 94°C for 5 min; 35 successful cycles of denaturation at 94°C for 45 sec, annealing at 52°C for 45 sec, and extension at 72°C for 45 sec; and the final extension at 72°C for 7 min.Primers were used for the detection of mecA gene, as shown in Staphylococcus aureus and negative control which was sterile distilled water were included during PCR running.All samples were aseptically collected and analyzed; positive control which was a well-known enterotoxin and mecA gene producing S. aureus was observed in 137 (34.25%) of the 400 food samples collected from different areas in Khartoum State. However, 126 (31.5%) of the 400 samples were identified as bacteria other than S. aureus, and 137 (34.25%) samples did not yield any growth  S. aureus isolates , while the rest of 82 isolates were negative. All isolates were negative for other enterotoxins gene (other than seb gene)    .S. aureus was observed in 30 (30%) of the 100 milk samples  samples showed no growth. All tested meat S. aureus isolates were susceptible to ciprofloxacin. The resistant rates of meat S. aureus isolates to gentamicin was 4 (13.3%), and it was higher than that identified to the antibiotics oxacillin and vancomycin with 2 (6.6%) and 5 (16.7%), respectively , while bacteria other than S. aureus represented 4%. However, none of the rest 76 (76%) showed any growth on the agar plate surface  were analyzed for the presence of bacterial pathogens. The study revealed that 24/100 (24%) of the fish samples had mination . Antibioibiotics . No enteibiotics .S. aureus, 26 (26%) were identified as bacteria others than S. aureus, and 11 (11%) showed no growth  and 87.3% (55/63 isolates), respectively, followed by oxacillin with a percentage of 84% (53/63). However, the least potent antibiotic was vancomycin with a percentage of 65% (41/63)  were identified as o growth . The ant (41/63) . The entch genes .S. aureus and MRSA and enterotoxin gene products were investigated in various food samples collected from Khartoum State markets . Identification of the bacteria isolated from the selected foods through conventional methods yielded a total of 137 (34.3%) S. aureus isolates. Similar reports on foods contaminated with S. aureus from Italy and India revealed much lower percentage yielding 17.1% [S. aureus in various foods revealed that the contamination levels with S. aureus reported were lower than those obtained in this study. On the other hand, a study in Greece reported 47.8% of northcentral and northeastern Greece foods, which have a much higher contamination level compared to those reported in this study [In this study, the prevalence of ng 17.1%  and 12.0ng 17.1% , respectS. aureus responsible for resistance to β-lactam antimicrobials was detected by using PCR, and we found that 15 (17%) S. aureus isolates were positive for the mecA gene while 69 (83%) were negative. Comparing our finding to previous studies results, we realized that most of the previous studies results showed a significantly higher prevalence than our finding, for example, in a study performed by Khayri in Makkah city, about 44.4% of his S. aureus isolates were positive for mecA gene [S. aureus isolates were positive for the mecA gene [In this study, resistance gene (mecA) of hile 69 8% were neS. aureus. Thirty (30%) samples were found to be contaminated with S. aureus. These findings highlight the high potential risk for consumers of meat and dairy products especially in the absence of strict hygienic and preventive measures to avoid Staphylococcus aureus enterotoxins (SEs) production in foods. In other comparative studies, similar results were reported from the USA where the prevalence of S. aureus in meat samples was 29.0% [aureus was 26.5% [S. aureus incidence was in 46.1% [S. aureus isolates was resistant to ciprofloxacin and 13.3% was resistant to gentamicin. In disagreement with our result, Das and Mazumder in India found that 16.66% of S. aureus meat isolates were resistant to ciprofloxacin [Staphylococcus aureus (VRSA) is a type of antibiotic-resistant S. aureus which have developed resistance and can no longer be treated with vancomycin. This study showed that 16.6% of the meat isolates were resistant to vancomycin, which suggests that the contamination may be coming from VRSA carrier's food handlers and processors; however, Das and Mazumder found that 3.33% of the isolates were resistant to vancomycin (VRSA) which is low compared with our findings [S. aureus (MRSA) strains have acquired a gene that makes them resistant to nearly all beta-lactam antibiotics; animal-adapted MRSA strains also exist. Although it is in small percentage, it has clinical importance and may cause serious problems to immunocompromised individuals as well as healthy ones (carriers). In this study, 6.6% of meat isolates were resistant to oxacillin; this finding was high compared to the results of van Loo et al. who found that 2.5% of S. aureus meat isolates were resistant to oxacillin [S. aureus isolates were resistant to oxacillin [In this study, one hundred raw meat samples were obtained from different supermarkets and restaurants in Khartoum State. These samples are examined for the presence of as 29.0% . Much loas 26.5% . Also, oin 46.1% . In our floxacin  and Pu efloxacin . Vancomyfindings . Methicixacillin  and low xacillin .S. aureus isolates. A similar study in Denmark demonstrated the presence of enterotoxin genes in only 0.2% of the isolates [S. aureus is estimated to be around 25% [S. aureus food isolates are not SEs producers; thus, a considerable research effort is still required for a better understanding of the interactions between S. aureus and the food matrix and of the mechanism of SEs production in foods stuffs [S. aureus-contaminated meat effectively. However, there is always the possibility of mutation at the level of the corresponding gene, leading to the absence of its detection. Therefore, a positive PCR shows the presence of the enterotoxin genes, but a negative PCR does not point to the absence of the corresponding operon because there are many types of enterotoxin genes and we determine only one type [Investigations through PCR technique on enterotoxin genes in this study showed the absence of enterotoxin genes in meat isolates . On the isolates , and Beround 25% . MoreoveS. aureus contamination in cheese sample in this study is higher than that reported in Iran and Japan where the reported contamination rates were 16% and 13.3%, respectively [Staphylococcus aureus was found on white cheese [S. aureus isolates to gentamicin and ciprofloxacin was 100%; these findings oppose those reported in the USA, where 75% of S. aureus isolates were resistant to gentamicin [S. aureus isolates were resistant to ciprofloxacin [S. aureus to oxacillin in this study was 5%, which was lower than that reported in Iraq which was 20% [In this study, one hundred white cheese samples collected from different retailers in Khartoum State to detect the rate of ectively , 27 and ectively . Howevere cheese . This mantamicin  and in Ifloxacin . The res was 20% . The sen was 20% .Staphylococcus aureus enterotoxins  genes among cheese isolates resulted in the detection of seb gene in 10% of the 20 isolates that was lower than the results obtained by Salheen in Sudan who reported that 20% of seb gene was detected in cheese samples [The molecular detection of  samples . The varS. aureus isolates showed 100% sensitivity to ciprofloxacin, which was in agreement with reports in Egypt [S. aureus isolates, which is similar to reports from Egypt [S. aureus isolated from fish samples showed a sensitivity rate of 92% to vancomycin, which is similar to those reported in Egypt (91%) [S. aureus isolates which was higher than that reported in Portugal (62%) [In this study, 108 fish samples were examined. The contamination rate of fish samples in this study was very low (22%) when compared with a previous report in Khartoum State (72%) , Egypt   and Portpt (91%) , respectpt (91%) , while opt (91%) , 42. Theal (62%) , while ial (62%) , 38. TheS. aureus isolates gave no band for all genes meaning that none of the all isolates possesses such gene in their genetic material; this finding agreed with those reported in Turkey and the USA [S. aureus isolates. However, a study in Tanzania reported that enterotoxins B and C genes were detected in 0.3% with the absence of enterotoxin A gene [The molecular detection of the enterotoxin's genes among fish  the USA , 43, when A gene , while an A gene . VariatiS. aureus was isolated in 63% of raw milk samples, which is relatively close to that reported in Brazil (68%) [S. aureus in this study is too high compared to reports from Sudan and Egypt where the levels were 30% and 24.8%, respectively [.S. aureus isolated from milk samples in this study were highly sensitive to ciprofloxacin (98.4%), which is close to a report from a study in Bangladesh (93.3%) [In this study, il (68%)  and Malail (68%) . Howeveril (68%)  and Egypil (68%) . The preil (68%) , 50. Morectively , 52.S. a (93.3%) . However (93.3%) , 55. The (93.3%)  and (100 (93.3%)  and high (93.3%) .S. aureus isolates were sensitive to gentamicin [S. aureus isolates are susceptible to gentamicin [S. aureus, and the result showed that 65% of the S. aureus isolates were susceptible to vancomycin; this finding was completely close to findings reported by Idbeis, in Basrha in Iraqe, and AL-Marsomy and Bendahou et al., in North Morocco, who mentioned that S. aureus isolated from raw milk and milk products showed sensitivity to vancomycin (100%) [S. aureus isolates tested for the presence of enterotoxins genes yielded negative results. Similar findings were reported in Hungary [While slightly similar to results obtained by Beyene in Ethiopia, Thaker et al. in India reported that 90% of ntamicin , 56. Repntamicin . In thisn (100%) –60. On tn (100%) , 61. Then (100%)  and lowen (100%) . All mil Hungary . The varStaphylococcus aureus with very low frequency of enterotoxin B gene in different kinds of selected food samples collected from Khartoum State. These findings elucidate the increased risk on public in Khartoum being affected by Staphylococcal food poisoning upon consumption of dairy or meat products prepared in unhygienic conditions that could lead to intoxication by Staphylococcus aureus enterotoxins.There was a relatively moderate prevalence of methicillin-resistant


Background: To evaluate the safety and neurological outcomes of therapeutic hypothermia to neonatal hypoxic-ischemic encephalopathy (HIE).Materials and Methods: Medical records of 61 neonates with moderate to severe HIE were retrospectively enrolled and divided into a therapeutic hypothermia group (n = 36) and conventional therapy group (n = 25).Results: No significant difference in the incidence of severe adverse events was found between the two groups. Minimum and maximum voltages of amplitude-integrated electroencephalography (aEEG) recording results showed statistically significant differences in therapeutic hypothermia group after 72 h. The neonatal behavioral neurological assessment (NBNA) on the 28th day after birth and Bayley Scales of Infant Development, second edition (BSID II) scores at 18 months old were significant higher in the therapeutic hypothermia group than the conventional therapy group.Conclusion: Therapeutic hypothermia for neonates with moderate to severe HIE improved the development of the nervous system in 0–18-month-old infants and showed a predominant role in reducing death and major neuron development-associated disabilities. Neonatal hypoxic-ischemic encephalopathy (HIE), mainly caused by perinatal asphyxia, commonly leads to brain damage which manifests as growth disorders, neuro-disability, and even death . The incRecently, a number of clinical trials and meta-analyses have indicated that therapeutic hypothermia confers benefits in HIE with reductions in mortality and adverse developmental outcomes in full-term newborns with moderate or severe HIE . It has The Shanghai Children’s Hospital Institutional Review Board approved this study. We performed a single-center, retrospective study of neonates admitted to the NICU of Shanghai Children’s Hospital who were diagnosed with moderate to severe HIE  to maintain rectal temperature at 33.5 ± 0.5°C within 10 h after birth. After 72 h of treatment, infants were rewarmed to rectal temperature of 37°C ± 0.5°C over 6–12 h (<0.5°C per hour). Temperature, respiration, heart rate, blood glucose, blood gas analysis, and electrolytes were closely monitored during therapeutic hypothermia. Once serious complications occurred  score on the 28th day after birth and Bayley Scales of Infant Development, second edition (BSID II) followed up to 18 months old were assessed.Early evaluation criteria of NBNA contain three characteristics: (1) scoring criteria: a total of 20 basic items, divided into 5 parts. Behavior abilities (6 items), passive muscle tension (4 items), active muscle tension (4 items), primitive reflex (3 items), and general evaluation (3 items). Score points have three degrees: 0 points, 1 point, 2 points, out of 40 points; the points method is based on the above 20 items with the choice of gurgling, red ball, talking face response, visual, and other sensibilities, and head erection and support with a total of five items, each score has three degrees: 0 points, 1 point, 2 points, with a full score of 10 points. (2) Criteria: NBNA basic project score < 35 points, suggesting the possibility of brain damage. (3) NBNA scores were performed on 98 subjects on the 28th day after birth, and were trained by specially trained professionals.For BSID II assessment, a mean (±SD) score of 100 ± 15 was normal on the Mental Development Index (MDI) and Psychomotor Developmental Index (PDI). Severe disability was defined as any of the following: a Bayley MDI score more than 2 SD below the mean score , a Gross Motor Function Classification System (GMFCS) grade level of 3 to 5, hearing impairment requiring hearing aids, or blindness. Moderate disability was defined as MDI score 1 to 2 SD below the mean score  and any of the following: a GMFCS level of 2, a hearing impairment with no amplification, or an active seizure disorder. Meanwhile the adverse effects of therapeutic hypothermia, severe disability and death were observed at the same time.t-test for differences between the two groups, and with chi-square test for the comparison of the sample rate. P < 0.05 was considered statistically significant.SPSS 17.0 was employed for statistical analysis. The data of normal distribution were expressed as mean ± standard deviation (x̄ ± s). The data were analyzed with A total of 61 newborns diagnosed with moderate to severe HIE were enrolled from January 2010 to December 2016 in the NICU of Shanghai Children’s Hospital, including 36 cases in the therapeutic hypothermia group and 25 cases in the conventional therapy group.p > 0.05).The baseline characteristics of the two groups are shown in Complications during the 72-h treatment and rewarming period for the therapeutic hypothermia group were recorded and compared with the conventional therapy group. There was no statistically significant difference in cold injury syndrome [0 (0.0%) vs. 0 (0.0%)], thrombocytopenia [5 (13.9%) vs. 4 (16.0%)], infection [7 (19.4%) vs. 5 (20.0%)], electrolyte disorders including: hypokalemia, hyperkalemia, hyponatremia, and hypocalcemia [14 (38.9%) vs. 10 (40.0%)], blood glucose disorder including: hypoglycemia and hyperglycemia [10 (27.8%) vs. 7 (28.0%)], and abnormal liver and kidney function [8 (22.2%) vs. 6 (24.0%)] between the therapeutic hypothermia group and conventional therapy group .p < 0.05] : (18.6 ± 2.5) vs. (22.4 ± 3.1), minimum voltage (μV): (6.5 ± 1.9) vs. (8.2 ± 2.6)]. However, there were significant differences in aEEG recording after 72 h of treatment  .The NBNA score of infants who were treated with therapeutic hypothermia was significantly increased compared with the infants in the conventional therapy group (39.1 ± 1.6 vs. 34.2 ± 2.1). The neurobehavioral development outcome was evaluated and followed up to 18 months old by standard BSID II. The MDI in the two groups were 96.5 ± 13.1 vs. 85.2 ± 10.7 respectively. The PDI in the two groups were 98.4 ± 15.7 vs. 86.3 ± 14.2, respectively. As can be seen from the above results, the MDI and PDI of infants in the therapeutic hypothermia group were significantly higher than that of the conventional therapy group . Furtherr = −0.1763, p = 0.3036)  , whereas the minimum voltage had a positive correlation with PDI  (The correlation between aEEG recording of HIE infants after therapeutic hypothermia and BSID II scores at 18 months old is shown in  0.3036) , while t 0.0379) . Likewis 0.0408) . The corHypoxic-ischemic encephalopathy is characterized as brain damage after encountering various neonatal hypoxic-ischemic injuries . Most cap > 0.05). The results suggested that therapeutic hypothermia was safe for newborns with moderate to severe neonatal HIE under the condition of closely monitoring and performing necessary laboratory tests during the treatment. Therapeutic hypothermia not only significantly decreased the incidence of serious disability and mortality, but also made nervous system prognosis better.Previous studies have reported several serious adverse events during therapeutic hypothermia, including apnea, coagulation disorder dysfunction, serious infection, hemorrhage or thrombosis, and arrhythmia . WhereasCompared with the conventional EEG, aEEG has the advantages of intuitive graphics, easy analysis, easy operation, and small levels of interference . The resA concern with any therapy that reduces mortality among infants at high risk of death and disability is the possibility of an increase in the number of infants who survive with disabilities. In our study there was no evidence to show that the moderate or severe disability rates increased at 18 to 22 months old among infants treated with therapeutic hypothermia.At present, it is emphasized that therapeutic hypothermia must be initialized within 6 h after birth, even during neonatal transport . StudiesBrain development and injury can be evaluated from different perspectives which include a clinical individual perspective  and cell electrophysiological perspective . Previous studies have shown that aEEG monitoring of neonates provided useful information to evaluate cerebral activity . The aEEIn conclusion, our study showed that therapeutic hypothermia with an aEEG monitor is effective and safe for newborns with moderate to severe HIE, and could significantly improve the prognosis according to the BSID II.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Institutional Review Board, Shanghai Children’s Hospital. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.BW, CC, and CY made substantial contributions to the conception and design, acquisition of data or analysis, and interpretation of data, and involved in drafting the manuscript or revising it critically for important intellectual content. CC and XG revised the manuscript and gave the final approval of the version to be published. All authors agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved and read and approved the final manuscript.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.