Enterobacter cloacae (CREC). The purpose of the current study was to assess in vitro antimicrobial and antibiofilm activities of VA against CREC. Here, minimum inhibitory concentrations (MIC) of VA against CREC was determined via gradient diffusion method. Furthermore, the antibacterial mode of VA against CREC was elucidated by measuring changes in intracellular adenosine triphosphate (ATP) concentration, intracellular pH (pHin), cell membrane potential and membrane integrity. In addition, antibiofilm formation of VA was measured by crystal violet assay and visualized with field emission scanning electron microscopy (FESEM) and confocal laser scanning microscopy (CLSM). The results showed that MIC of VA against E. cloacae was 600 μg/mL. VA was capable of inhibiting the growth of CREC and destroying the cell membrane integrity of CREC, as confirmed by the decrease of intracellular ATP concentration, pHin and membrane potential as well as distinctive variation in cellular morphology. Moreover, crystal violet staining, FESEM and CLSM results indicated that VA displayed robust inhibitory effects on biofilm formation of CREC and inactivated biofilm-related CREC cells. These findings revealed that VA exhibits potent antibacterial activity against CREC, and thus has potential to be exploited as a natural preservative to control the CREC associated infections.Vanillic acid (VA) is a flavoring agent found in edible plants and fruits. Few recent studies exhibited robust antibacterial activity of VA against several pathogen microorganisms. However, little was reported about the effect of VA on carbapenem-resistant  Enterobacter cloacae (E. cloacae) is a common gram-negative, rod-shaped bacterium belonging to the family Enterobacteriaceae and can be frequently detected from human and animal excrement. E. cloacae has also been isolated from food processing plants, rice, seafoods and meat products, which can bring out food spoilage  was applied to determine the changes of bacterial membrane potential. Bacteria in the logarithmic phase of growth were harvested by centrifugation at 4000× g for 10 min, rinsed twice with 10 mM phosphate-buffered saline , and the bacterial concentration was adjusted to 1 × 105 CFU/mL. The bacterial suspensions were treated with VA  at 37 °C for 2 h and then incubated with DiBAC4(3) at 25 °C in the dark for 10 min as well as then washed with PBS. Finally, fluorescence was calculated on a fluorescence microplate reader.Membrane potential was determined according to the method described by Wang et al. with minor modifications . Brieflyg for 5 min, and resuspended with equivalent volumes of 0.01 M PBS. After cell suspensions were incubated with the mixture of cFDA SE and propidium iodide (PI) at 37 °C for 15 min, cells were collected and washed with 100 mM PBS to remove excess dyes.Cell membrane integrity of CREC cells was assessed by confocal laser scanning microscopy  according to a modified method of Liu et al. . The midg, 8 min) and washed twice with 0.85% NaCl. Following removal of the supernatant, cell pellets were fixed with 2.5% glutaraldehyde at 4 °C for 12 h, and there after dehydrated by 20%, 50%, 70%, 80%, 90% and 100% alcohol for 15 min and finally embedded in resin. Ultrathin samples were cut on ultra-microtome, stained with uranyl acetate, and observed by TEM.TEM  observation was performed as described by Joung et al. with some modifications . ExponenMorphology changes of the bacterial after treatment with VA at different concentrations were observed using a field emission scanning electron microscopy  according to previous methods . These l570) normalized with cell growth (OD630).The effect of VA on biofilm formation of CREC was tested quantitatively in 96-well plates by modified crystal violet staining assay method as described by Dasagrandhi et al. with some modifications . FollowiEffects of VA on the biofilm formation were visually observed by FESEM and CLSM with minor modifications . The sliIn addition, CREC cells grown for 12 h on the slides were precultured and then exposed to VA of different concentrations  for further culture of 24 h. After incubation, the slides of cells were washed three times with 0.85% NaCl. cFDA SE were mixed thoroughly with CREC cells grown well on the slides for direct visual observation of biofilm formation, which were incubated at 25 °C for 15 min. The bacterial biofilm was examined by CLSM, where the fluorescence intensity of cells grown on the slides was measured at excitation/emission wavelengths of 485/542 nm for cFDA SE.The damage of CREC cells within biofilm was observed via CLSM according to a previous study . CREC cep ≤ 0.01.All experiments were performed independently three times, and each biological replicate included two technical replicates. Results were analyzed utilizing using Origin 8.5 Statistics. All data were expressed as mean values and standard deviation (SD). Differences are considered significant at In summary, VA was effective in inactivating both CREC cells and biofilm formation. VA could induce in cell lysis resulting in the cell membrane damage and leakage of intracellular components of CREC cells. Additionally, VA at 2MIC can inactivate biofilm CREC cells, and thus has potential use as natural antibacterial agents to control CREC contamination in the food industry.


To describe the epidemiological aspects of HIV infection and AIDS among indigenous peoples of the state of Mato Grosso do Sul, Brazil.Distrito Sanitário Especial Indígena  Mato Grosso do Sul between 2001 and 2014, based on three secondary databases. Annual rates of HIV and AIDS detection and prevalence were calculated, considering case distribution according to village, Health Base Pole and sociodemographic variables. Accumulated rates of detection, mortality and case fatality were calculated by ethnic group and for the Health Base Pole with the highest number of cases. This is a descriptive epidemiological study on the occurrence and distribution of HIV infection and AIDS in the indigenous population assisted by the  The HIV detection rate fluctuated between 0.0 and 18.0/100 thousand people in the study period. For AIDS, there was no notification before 2007, but in 2012 its rate reached 16.6/100 thousand. HIV prevalence grew between 2001 and 2011, and it continuously grew for AIDS starting from 2007. The highest HIV detection rates occurred among Guarani peoples (167.1/100 thousand) and for AIDS, among the Kaiowá peoples (79.3/100 thousand); mortality and fatality rates were higher among the Kaiowá. Regarding the Dourados Health Base Pole, the AIDS detection rate increased, and the mortality and fatality rates decreased. HIV infection and AIDS have been increasing among indigenous peoples, with distribution of the disease mainly in the Health Base Poles of the southern region of the state, where greater economic and social vulnerability are also observed. The endemic character of HIV and AIDS can become epidemic in some years given the existence of cases in other villages in the state. Its occurrence among the Guarani and Kaiowá populations indicates the need for expanded diagnosis, access to treatment and prevention measures. In Brazil, from 1980 to June 2017, 882,810 cases of AIDS were recordedb . Between 2006 and 2016, there was a trend towards the stabilization of the AIDS detection rate, considering a 18.5/100 thousand inhabitants mean in 2016. However, in the state of Mato Grosso do Sul, an 8.2% growth occurred in the period, represented by a 19.8/100 thousand inhabitants rate in 2016, the highest in the Midwest regionb .Acquired immunodeficiency syndrome (AIDS) has already affected millions of people worldwide. By 2015, about 36.7 million people were living with the virus2 . Information from countries such as the United States, Canada, New Zealand and Australia indicates a significant increase in the HIV detection rate among indigenous peoples in recent decades5 . In general, this rate is higher among indigenous individuals than non-indigenous, indicating health inequities in the access to diagnosis and treatment of the disease5 .The impact of AIDS among indigenous populations is still little known. However, studies have highlighted the greater vulnerability of these population groups to HIV transmission, related to bad living conditions, lower socioeconomic and educational level, social exclusion and difficulty in accessing health servicesb . However, the impact of AIDS on indigenous peoples cannot be characterized by absolute numbers and/or rates only. The sociocultural, economic, demographic, and geographic dimensions of each indigenous people, associated with the process of interaction with non-indigenous society, expose them to unique risk factors that lead to greater vulnerability when compared with other Brazilian population groups.The first case of AIDS in indigenous populations in Brazil was recorded in 1987, and only 33 cases were known until 1999. This number increased to 1,042 between 2000 and June 2017Subsistema de Atenção à Saúde Indígena , within the scope of the Unified Health System d .The provision of health services for indigenous peoples also has repercussions in this scenario. In recent Brazilian history, the health care of indigenous peoples gained strength in 1999 with the creation of the Considering the need for investigations that fill the gaps related to the occurrence of HIV and AIDS among indigenous peoples, and that can subsidize the formulation of appropriate care policies directed to this population, this study aimed to describe the epidemiological aspects of these cases among the indigenous peoples of the state of Mato Grosso do Sul, from 2001 to 2014.Distrito Sanitário Especial Indígena Mato Grosso do Sul , from 2001 to 2014. Mato Grosso do Sul has the second largest indigenous population in Brazil, with 73,295 self-declared individualse . The DSEI-MS has 14 Health Base Poles, which are the first reference for the multidisciplinary teams of indigenous health (EMSI) that operate in the villages and care for 70,032 people from eight ethnic groups  distributed in 75 villages and 26 camps, located in 31 of the 79 municipalities of the statef .This is a descriptive study on the occurrence and distribution of HIV and AIDS cases on the indigenous population of the state of Mato Grosso do Sul, assisted by the Sistema de Informação de Agravos de Notificação (Information System of Notifiable Diseases – Sinan), being selected by the indigenous race/color variable, who lived in villages and camps of the state. The exclusion criteria were duplicate cases, indigenous people living in villages/urban areas not assisted by DSEI-MS, and children exposed to HIV during pregnancy.This study comprised individuals with diagnoses of HIV infection from the records of daily evaluations made by the EMSI, and of AIDS from the Sistema de Informação de Atenção à Saúde Indígena . The place of residence was considered as the one registered at the time of the HIV diagnosis by the EMSI or the submitted to Sinan for AIDS cases. Sociodemographic variables  were obtained from Siasi, as well as the population bases used for calculating the rates described below.The data recorded by the EMSI during the study period were obtained from June 2014 to January 2015 by manually linking the name, date of birth and name of the mother to three databases: 1. records from daily evaluations made by EMSI; 2. the Sinan database; 3. The number of newly recorded cases of each was used to calculate the annual rates of HIV and AIDS detection. People directly diagnosed with AIDS were only considered for the AIDS detection rate. To include people diagnosed in areas unassisted by DSEI-MS, the territory to which the person moved to was considered, as well as the year in which it occurred.The number of old and new HIV cases of each year was used to calculate HIV prevalence. Deaths, cases that evolved to AIDS, and people who moved from the areas served by DSEI-MS were excluded. The number of old cases and the new diagnoses was used to calculate AIDS prevalence, in addition to those that evolved to AIDS in that year. Deaths and people who moved from the areas served by DSEI-MS were excluded.® statistical program, version 24.0, considering a 5% significance level.The analysis of the association between the years and the detection rate of HIV and AIDS, as well as the prevalence, was performed using Pearson’s chi-square test with Bonferroni correction when necessary, using the IBM SPSSThe other results were presented as descriptive statistics. The spatial distribution of HIV and AIDS cases in the area covered by DSEI-MS was determined according to the place of residence  at the time of diagnosis. HIV and AIDS detection rates, as well as accumulated AIDS mortality and fatality for the whole period were estimated, considering the total population of each ethnic group in 2014. The accumulated rates were also calculated for the three Health Base Poles that concentrated the most diagnoses, divided in two periods  and .Comissão Nacional de Ética em Pesquisa (Brazilian Research Ethics Commission – CONEP) under Opinion 707.439/2014.This study was approved by the HIV infection detection rates showed annual fluctuations in the study period, with values ranging from zero to 18.0/100 thousand people. The highest rates were recorded in 2006 (17.8/100 thousand) and 2011 (18.0/100 thousand). Regarding AIDS cases, there were no records of the disease between 2001 and 2006. From 2007, an increase was observed until 2012, when the detection rate reached 16.6/100 thousand. In 2012 and 2013, AIDS detection rates were higher than HIV detection rates   .Annual HIV prevalence rates increased in the period from 2001 to 2011, and decreased from 2011 onward, decreasing from 51.2/100 thousand to 39.6/100 thousand in 2014. For AIDS, the prevalence rate increased continuously starting from 2007, gradually approaching the HIV infection prevalence after 2012   .During the study period, 97 HIV infection cases and 50 AIDS cases were recorded, involving 103 people, of whom 6 were directly diagnosed with AIDS. The distribution of cases was quite uneven despite HIV being found in 80% of the Health Base Poles and AIDS in 64% of them. Jointly, the Centers of Dourados, Amambai and Iguatemi concentrated 69% of HIV infection cases and 68% AIDS cases. The Dourados Health Base Pole alone accounted for 34% of HIV infection cases. The Jaguapirú, Amambai, Taquapery and Porto Lindo villages recorded 52% of the AIDS cases, 20% of which were in the Jaguapiru village    .Regarding the accumulated detection rates of HIV and AIDS by Health Base Pole, there were some ordinal differences in distribution. The Brasilândia Health Base Pole had the highest HIV detection rate , but its population size must be considered. The following Health Base Poles were Dourados (217.3/100 thousand), Iguatemi (215.4/100 thousand) and Amambai (177.6/100 thousand). For the AIDS cases, the detection rate was higher for the Health Base Pole of Amambai (115.8/100 thousand), Tacuru (99.2/100 thousand) and Iguatemi (97.9/100 thousand), leaving the Dourados Base out of the first three places   .The age group with the highest incidence of HIV and AIDS infection was 20 to 39 years old, with 43 (44.3%) and 22 (44.0%) cases, respectively. There was statistical significance for HIV cases, for a 0.040 p-value. In the distribution by sex, most HIV and AIDS cases occurred among women, corresponding to 60 (61.9%) and 30 (60%) cases, respectively. We note that HIV infection occurred earlier in women than in men, given that in the 10-19 age group the ratio between the sexes was approximately nine women for each man   .The highest percentages of HIV and AIDS infection were found in indigenous people with incomplete elementary education, represented by 45 (46.4%) and 27 (54.0%), respectively, followed by illiterates, represented by 20 (20.6%) and 13 cases (26.0%), respectively, with statistical significance for HIV cases (p = 0.016). Still regarding schooling, we identified a high percentage of records without such information – 20.6% for HIV and 12.0% for AIDS   .Of the eight ethnic groups served by the DSEI-MS, five had registered cases: Kaiowá, Terena, Guarani, Kadwéu and Ofaié, as well as non-indigenous individuals living in the villages. The Kaiowá ethnic group had the highest concentration of HIV infection, with 50 cases (51.5%), followed by the Terena ethnic group with 22 cases (22.7%). The same ethnic groups were also predominant for AIDS cases, with 27 (54.0%) and 15 cases (30.0%), respectively   .Although the HIV and AIDS detection rates were higher for the Guarani and Terena ethnic groups, we must highlight the Ofaié ethnic group, whose rates were considerably higher than the other when considering their small population size. Mortality and fatality rates were also higher for the Kaiowá when compared to the other ethnic groups. Moreover, deaths were recorded only for the Guarani and Terena ethnic groups. Although the Terena had the lowest mortality rate, they had the second highest fatality rate   .Comparisons of detection, mortality and fatality rates between the two analyzed periods (2001 to 2007 and 2008 to 2014) showed that the AIDS detection rate increased more than that of HIV infection. Furthermore, the mortality rate by AIDS increased, whereas the fatality decreased. Of the Health Base Poles with highest detection rates of HIV and AIDS cases, Iguatemi was the only one that showed a decrease in both (HIV and AIDS). In contrast, the Amambai Center presented increased detection rates for both HIV and AIDS. The AIDS detection rate increased in the Dourados Center (25.4/100 thousand for 72.4/100 thousand) whereas its mortality and fatality decreased   .This study showed the evolution of the detection rates of HIV infection and AIDS in the indigenous population of Mato Grosso do Sul. Women in the 20 to 39 years age group, with lower schooling and of the Kaiowá ethnic group presented the highest HIV and AIDS infection percentages. The Kaiowá, Terena and Guarani ethnic groups were the most exposed one, with records of high HIV infection and AIDS rates. These results also contributed to the high mortality and fatality rates in these groups. The Health Base Poles of Amambai, Dourados and Iguatemi, located in the southern region of the state, concentrated around 70% of cases of HIV infection and AIDS.Oscillations in detection rates in the study period may be more strongly associated with the diagnostic ability of services than to the epidemiological behavior of the virus transmission or disease burden on the population. These must be interpreted as a warning sign for the health care services for the state’s indigenous peoples.7 . The historical context of the contact of these peoples with the non-indigenous society, their geographic location along a border, the proximity and ease of access between villages and urban centers – something common in Mato Grosso do Sul’s villages –, associated with social problems such as unemployment, violence, exploitation and sexual abuse14 indicate a situation of great vulnerability that may help to understand the concentration of two-thirds of the cases identified in this study between these two ethnic groups, especially in the villages belonging to the Dourados, Amambai and Iguatemi Health Base Poles.The Guarani and Kaiowá have a very complex health picture, with high prevalence of infectious diseases, high infant mortality and malnutrition, as well as the increasing prevalence of chronic non-communicable diseases9 – hinders the regular access to health services and diagnosis. Situations of intense inter-ethnic contact and marginalization in the access to health services are also risk factors for the circulation and spread of HIV. Socioeconomic, environmental, and cultural determinants interfere with the intensity of disease spread, making prevention and control activities complex, as evidenced in a study on the introduction of AIDS in the Xokleng people of Santa Catarina17.Furthermore, the mobility between villages – even outside the Brazilian territory since the Guarani and Kaiowá territories extend to Paraguay19 , including for indigenous peoples. Studies have shown that schooling is also directly associated with the survival of patients with HIV and AIDS, having a protective effect against most causes of death21 .In this study, the population with the greatest vulnerability to the spread of HIV and AIDS were young people, especially women with low schooling level. Unfavorable socioeconomic conditions are associated with greater exposure to risk factors. Schooling is a social status marker and has effects on the access to information and knowledge about disease prevention methods and its treatment19 . Another study on sexually transmitted infections, also conducted on the indigenous population of Mato Grosso do Sul between 2001 and 2005, first identified the prevalence of AIDS among men up to 2002, and in the following years, the number of cases between sexes was matched22 . However, these results do not allow us to affirm that a “feminization” of HIV and AIDS cases has occurred among indigenous peoples in Mato Grosso do Sul because, as has been observed in Brazil23 , such rates may be related to the greater coverage of women’s health provided by the Programa Estadual de Proteção à Gestante (State Protection Program for Pregnant Women), which conducts HIV screening tests during pregnancy.The findings of this study differ from those observed among the indigenous peoples of Peru and Bolivia, whose HIV infection/AIDS ratio among men and women was 3:1 and 5:3, respectively24 . The predominance of AIDS cases diagnosed in the 20 to 39 years and 40 to 60 years age groups was also found in the Brazilian scenariob and in a study conducted among the indigenous peoples of Bolivia and Peru, which found one in two cases among 20 and 39 years19 .On the other hand, the limitations of the diagnosis of HIV infection in the male indigenous population may be associated with the low coverage of diagnostic tests, absence of men in the villages for work-related reasons, and reduced male demand for health services. The low diagnostic coverage favors the spread of the infection since ignorance about the disease leads to the continuation of sexual practices without prevention. The gradual inclusion of rapid HIV testing in villages as of 2012 may have contributed to the resumption in the growth of the HIV detection rate in the last two years of the study period since it led to increased diagnostic coverage, as observed in other DSEIs across BrazilThe observation of mortality and fatality rates among the Kaiowá is relevant for the organization of health services since it indicates that this ethnic group has less access to antiretroviral therapy. The decrease in mortality and fatality rates in the Dourados Base in the second period suggests a greater structuring of health services in the villages, and articulation with specialized care services (SAE) for the treatment of indigenous individuals with AIDS. The differentiated behavior of the HIV and AIDS rates in each of the Health Base Pole shows the need for the separate evaluation of health services, given that these present different structuring forms to address the problem.The main limitations of this study are related to the use of secondary data sources, which compromise the quality of information. The deficiency in the completeness of the variable indigenous race/skin color in Sinan and the underreporting of HIV and AIDS cases in indigenous peoples are factors that influence the number of identified cases and, consequently, information coverage.g , and in 2014 for the general populationh . Underreporting and incompleteness of the variable race/skin color data make it difficult to characterize health inequity situations, and to establish strategies for coping with such inequities26 .This study used daily EMSI records as a data source to improve the coverage of HIV infection cases, since compulsory notification was only included in Sinan in 2005 for pregnant womenDespite these limitations, this study has brought benefits by addressing a disease that is not very well known in the epidemiology of indigenous peoples in Brazil, which impacts on the morbidity and mortality profile, and is related to the emergence of sexually transmitted infections and its association with social and cultural changes resulting from the process of interaction with the non-indigenous society.27 and the articulation between indigenous and non-indigenous health systems28 . Knowledge of logistical difficulties, population distribution and more vulnerable areas help to define strategies to expand the diagnostic coverage and facilitate the access to treatment29 . In this sense, the training of human resources to work in intercultural contexts is fundamental to increase response capacity Sasi-SUS in disease prevention and treatment actions.The challenge of working with sexually transmitted diseases in an inter-ethnic context implies the need to comprehend sexuality values inherent in each indigenous groupThis study showed that the endemic character of HIV and AIDS – with prevalence of HIV infection in the southern region of the state –, may become epidemic in some years since there are cases in other indigenous areas of Mato Grosso do Sul. Among the crucial intervention measures to address the problem, we can cite the expansion of diagnostic coverage, the guarantee of access to treatment, and measures to prevent HIV transmission. a . No Brasil, de 1980 até junho de 2017 foram contabilizados 882.810 casos de aidsb . Entre 2006 e 2016, observou-se no país uma tendência de estabilização da taxa de detecção de aids, com uma média de 18,5/100 mil habitantes em 2016. Contudo, no estado de Mato Grosso do Sul, houve crescimento de 8,2% no período, representado por uma taxa de 19,8/100 mil habitantes em 2016, a maior da região Centro-Oesteb .A síndrome da imunodeficiência adquirida (aids) já acometeu milhões de pessoas em todo o mundo. Em 2015, cerca de 36,7 milhões viviam com o vírus2 . As informações oriundas de países como Estados Unidos da América, Canadá, Nova Zelândia e Austrália indicam um aumento expressivo da taxa de detecção de HIV entre os povos indígenas nas últimas décadas3 . Em geral, essa taxa é maior entre indígenas do que entre não indígenas, o que indica as iniquidades em saúde, no que diz respeito ao acesso ao diagnóstico e tratamento da doença5 .Entre os povos indígenas, o impacto da aids ainda é pouco conhecido. Estudos, entretanto, têm destacado a maior vulnerabilidade desses grupos populacionais à transmissão do HIV, relacionada às piores condições de vida, menor nível socioeconômico, educacional, exclusão social e dificuldade de acesso aos serviços de saúdeb . Contudo, o impacto da aids em povos indígenas não pode ser caracterizado apenas por números absolutos e/ou taxas. As dimensões socioculturais, econômicas, demográficas, geográficas de cada povo indígena, associadas ao processo de interação com a sociedade não indígena, os expõem a fatores de risco singulares que levam à maior vulnerabilidade em relação aos demais grupos populacionais brasileirosc .No Brasil, o primeiro caso de aids em indígenas foi registrado em 1987, e até 1999 conheciam-se somente 33 casos. Já entre 2000 e junho de 2017, o número aumentou para 1.042d .A oferta de serviços de saúde para os indígenas também repercute neste cenário. No Brasil, em seu histórico recente, a atenção à saúde dos povos indígenas ganhou força em 1999 com a criação do Subsistema de Atenção à Saúde Indígena, no âmbito do Sistema Único de Saúde (Sasi-SUS)Considerando a necessidade de investigações que supram as lacunas relacionadas à ocorrência de HIV e aids entre indígenas e que possam subsidiar a formulação de políticas de assistência qualificadas direcionadas a essa população, este estudo teve por objetivo descrever os aspectos epidemiológicos desses casos entre os povos indígenas do estado de Mato Grosso do Sul, no período de 2001 a 2014.e . O DSEI-MS possui 14 Polos Base, primeira referência para as equipes multidisciplinares de saúde indígena (EMSI) que atuam nas aldeias e atendem 70.032 pessoas de oito etnias  distribuídas em 75 aldeias e 26 acampamentos, localizados em 31 dos 79 municípios do estadof .Estudo descritivo sobre a ocorrência e distribuição dos casos de infecção pelo HIV e aids na população indígena do estado de Mato Grosso do Sul assistida pelo Distrito Sanitário Especial Indígena Mato Grosso do Sul (DSEI-MS), no período de 2001 a 2014. Mato Grosso do Sul tem a segunda maior população indígena do Brasil, com 73.295 pessoas autodeclaradasNeste estudo, foram incluídos indivíduos com diagnósticos de infecção pelo HIV, oriundos dos registros dos atendimentos diários das EMSI, e de aids, provenientes do Sistema de Informação de Agravos de Notificação (Sinan), selecionados pela variável raça/cor indígena, que residiam em aldeias e acampamentos indígenas do estado. Excluíram-se os casos duplicados, os indígenas residentes em aldeias/áreas urbanas não assistidas pelo DSEI-MS e as crianças expostas ao HIV na gestação.Os dados registrados pelas EMSI no decorrer do período do estudo foram obtidos de junho de 2014 a janeiro de 2015 por meio da vinculação manual do nome, data de nascimento e nome da mãe entre três bases de dados: 1. registros provenientes dos atendimentos diários das EMSI; 2. banco de dados do Sinan; 3. Sistema de Informação de Atenção à Saúde Indígena (Siasi). Considerou-se local de residência aquele registrado no momento do diagnóstico de HIV pela EMSI ou o lançado no Sinan, para os casos de aids. As variáveis sociodemográficas  foram obtidas no Siasi, assim como as bases populacionais utilizadas para os cálculos das taxas descritas em seguida.Para calcular as taxas anuais de detecção de HIV e aids, utilizou-se o número de casos novos registrados de cada um. As pessoas diagnosticadas diretamente com aids foram consideradas somente para a taxa de detecção de aids. Para inclusão das pessoas diagnosticadas em áreas não assistidas pelo DSEI-MS, considerou-se o território para o qual a pessoa se mudou e o ano em que isso ocorreu.Para o cálculo da prevalência de HIV, utilizou-se o número de casos antigos e novos de HIV de cada ano e excluíram-se os óbitos, os que evoluíram para aids e as pessoas que se mudaram das áreas atendidas pelo DSEI-MS. Para o cálculo da prevalência da aids, utilizou-se o número de casos antigos e os novos diagnósticos, mais os que evoluíram para aids naquele ano, excluídos os óbitos e pessoas que se mudaram das áreas em que o DSEI-MS prestava assistência.®, versão 24.0, com um nível de significância de 5%.A análise da associação entre os anos e a taxa de detecção de HIV e de aids, assim como as prevalências, foi realizada pelo teste do qui-quadrado de Pearson, com correção de Bonferroni, quando necessário, utilizando o programa estatístico IBM SPSSOs demais resultados foram apresentados na forma de estatística descritiva. A distribuição espacial dos casos de HIV e aids na área de abrangência do DSEI-MS foi determinada segundo local de residência  no momento do diagnóstico. Calcularam-se também as taxas de detecção de HIV e de aids, assim como de mortalidade e letalidade por aids acumuladas referentes a todo o período, considerando a população total de cada etnia no ano de 2014. As taxas acumuladas também foram calculadas para os três Polos Base que concentraram a maioria dos diagnósticos, divididas em dois períodos  e .O estudo recebeu aprovação da Comissão Nacional de Ética em Pesquisa (Conep) sob o Parecer 707.439/2014.As taxas de detecção de infecção pelo HIV apresentaram flutuações anuais no período do estudo, com valores que foram de zero até 18,0/100 mil pessoas. As maiores taxas foram registradas em 2006  e 2011 . Em relação aos casos de aids, não houve registros da doença entre 2001 e 2006. A partir de 2007, observou-se um aumento até 2012, quando a taxa de detecção chegou a 16,6/100 mil. Em 2012 e 2013, as taxas de detecção de aids foram superiores às de HIV   .As taxas anuais de prevalência de HIV apresentaram crescimento no período de 2001 a 2011 e uma diminuição a partir de 2011, caindo de 51,2/100 mil para 39,6/100 mil em 2014. Para a aids, a taxa de prevalência apresentou aumento contínuo a partir de 2007, aproximando-se gradativamente da prevalência de infecção pelo HIV depois de 2012   .No período do estudo, registraram-se 97 casos de infecção pelo HIV e 50 de aids, envolvendo 103 pessoas, das quais 6 foram diagnosticadas diretamente com aids. Apesar de o HIV estar presente em 80% dos Polos Base e a aids em 64% deles, a distribuição dos casos foi bastante desigual. Os Polos de Dourados, Amambai e Iguatemi concentraram juntos 69% dos casos de infecção por HIV e 68% de aids. O Polo Base de Dourados sozinho registrou 34% dos casos de infecção pelo HIV. As aldeias Jaguapirú, Amambai, Taquapery e Porto Lindo registraram 52% dos casos de aids, 20% dos quais estavam na aldeia Jaguapiru (Polo Base de Dourados)   .Em relação às taxas de detecção de HIV e aids acumuladas por Polo Base, observaram-se algumas diferenças ordinais na distribuição. O Polo Base de Brasilândia obteve a maior taxa de detecção de HIV , mas é necessário considerar seu tamanho populacional. Em seguida vieram os Polos de Dourados , Iguatemi  e Amambai . Para os casos de aids, a taxa de detecção foi maior para os Polos de Amambai , Tacuru  e Iguatemi , deixando o Polo de Dourados fora dos três primeiros colocados   .A faixa etária de maior ocorrência de infecção pelo HIV e aids foi de 20 a 39 anos, com 43  e 22  casos, respectivamente. Houve significância estatística para os casos de HIV, cujo valor de p foi 0,040. Na distribuição por sexo, verificou-se que a maior parte dos casos de HIV e aids ocorreu entre as mulheres, correspondendo a 60  e 30 (60%) casos, nessa ordem. Nota-se que a infecção pelo HIV ocorreu mais precocemente no sexo feminino do que no masculino, visto que, na faixa etária de 10 a 19 anos, a razão entre os sexos foi de aproximadamente nove mulheres para cada homem   .Os maiores percentuais de infecção pelo HIV e aids foram encontrados em indígenas com ensino fundamental incompleto, representados por 45  e 27 casos , respectivamente, seguidos pelos analfabetos, representados por 20  e 13 casos , respectivamente, com significância estatística  para os casos de HIV. Ainda sobre a escolaridade, identificou-se um elevado percentual de registros sem informação – 20,6% para HIV e 12,0% para aids   .Das oito etnias atendidas pelo DSEI-MS, cinco tiveram casos registrados: Kaiowá, Terena, Guarani, Kadwéu e Ofaié, além de não indígenas residentes em aldeias. A etnia Kaiowá apresentou maior concentração de infecção pelo HIV, com 50 casos , seguida da etnia Terena, com 22 casos . Para os casos de aids, também observou-se predomínio das mesmas etnias, com 27  e 15 casos , respectivamente   .Ainda que as taxas de detecção de HIV e aids tenham sido maiores para as etnias Guarani e Terena, merece destaque a etnia Ofaié, cujas taxas foram consideravelmente superiores às anteriores, devido a sua pequena representatividade populacional. As taxas de mortalidade e letalidade foram igualmente superiores para os Kaiowá quando comparadas às de outras etnias. Além dessa, óbitos foram registrados apenas para as etnias Guarani e Terena. Apesar de os Terena terem apresentado a menor taxa de mortalidade, tiveram a segunda maior letalidade   .As comparações das taxas de detecção, de mortalidade e letalidade entre os dois períodos analisados (2001 a 2007 e 2008 a 2014) indicaram que a taxa de detecção de aids aumentou mais que a de infecção pelo HIV. Além disso, houve um aumento na taxa de mortalidade por aids e uma diminuição na letalidade. Dos Polos Base com maior taxa de detecção de casos de HIV e aids, Iguatemi foi o único que apresentou diminuição de ambas as taxas de detecção (HIV e aids). Em contrapartida, o Polo de Amambai mostrou aumento das taxas de detecção, tanto de HIV como de aids. Para o Polo de Dourados, observou-se elevação da taxa de detecção de aids  e, por outro lado, diminuição de suas taxas de mortalidade e letalidade   .O presente estudo demonstrou evolução das taxas de detecção de infecção pelo HIV e aids na população indígena de Mato Grosso do Sul. As mulheres na faixa etária de 20 a 39 anos, com menor escolaridade e da etnia Kaiowá apresentaram maiores percentuais de infecção pelo HIV e aids. As etnias Kaiowá, Terena e Guarani foram as mais expostas, com registros de altas taxas de infecção pelo HIV e aids. Esses resultados contribuíram, também, para as elevadas taxas de mortalidade e letalidade nesses grupos. Os Polos Base de Amambai, Dourados e Iguatemi, na região sul do estado, concentraram em torno de 70% dos casos de infecção pelo HIV e de aids.As oscilações nas taxas de detecção no período podem estar mais fortemente associadas à capacidade dos serviços para realizar diagnósticos e menos ao comportamento epidemiológico da transmissão do vírus ou à carga da doença na população. Elas devem ser encaradas como um sinal de alerta para os serviços de atenção à saúde dos povos indígenas do estado.7 . O contexto histórico de contato desses povos com a sociedade não indígena, a sua localização geográfica em uma faixa de fronteira, a proximidade e facilidade de acesso entre as aldeias e os centros urbanos, muito comuns nas aldeias do Mato Grosso do Sul, associados aos problemas sociais como desemprego, violência, exploração e abuso sexual14 , indicam uma situação de grande vulnerabilidade que pode ajudar a entender a concentração de dois terços dos casos identificados neste estudo entre essas duas etnias, em especial nas aldeias pertencentes aos Polos Base de Dourados, Amambai e Iguatemi.Os Guarani e Kaiowá apresentam um quadro de saúde bastante complexo, com alta prevalência de doenças infecciosas, elevada mortalidade infantil e desnutrição, juntamente com a prevalência crescente de doenças crônicas não transmissíveis9 , dificulta o acesso regular aos serviços de saúde e o diagnóstico. Situações de intenso contato interétnico e a marginalização no acesso aos serviços de saúde também constituem fatores de risco para a circulação e propagação do HIV. Os determinantes socioeconômicos, ambientais e culturais interferem na intensidade de propagação da doença e tornam complexas as atividades de prevenção e controle, como evidenciado em estudo sobre a introdução da aids no povo Xokleng de Santa Catarina17.Além disso, a mobilidade entre aldeias, inclusive para fora do território nacional, uma vez que o território Guarani e Kaiowá se estende para o Paraguai19 , inclusive para os povos indígenas. Estudos demonstraram que a escolaridade também está diretamente associada à sobrevida de pacientes com HIV e aids, tendo um efeito protetor contra a maioria das causas de óbito21 .No presente estudo, a população com maior situação de vulnerabilidade à propagação do HIV e aids foi a de jovens, principalmente as mulheres e com baixa escolaridade. Condições socioeconômicas desfavoráveis estão associadas à maior exposição aos fatores de risco. A escolaridade é um marcador de condição social e repercute no acesso à informação e no conhecimento de métodos de prevenção da doença e de seu tratamento19 . Já outro estudo sobre infecções sexualmente transmissíveis também realizado com a população indígena de Mato Grosso do Sul entre 2001 e 2005 identificou primeiramente o predomínio de aids entre os homens até 2002, e nos anos seguintes equiparação de casos entre os sexos22 . Entretanto, tais resultados não permitem afirmar que ocorreu uma “feminilização” dos casos de HIV e aids entre indígenas no Mato Grosso do Sul, como vem sendo observado no Brasil23 , pois podem estar relacionados à maior cobertura da assistência às mulheres proporcionada pelo Programa Estadual de Proteção à Gestante, que realiza exames de detecção de HIV na gestação.Os achados do presente estudo diferem do observado entre os povos indígenas do Peru e Bolívia, cuja razão de infecção pelo HIV/aids entre homens e mulheres foi de 3:1 e 5:3, respectivamente24 . A predominância de casos de aids diagnosticados na faixa etária de 20 a 39 anos e de 40 a 60 anos também foi verificada no cenário nacionalb e em estudo conduzido entre os povos indígenas da Bolívia e do Peru, que observou um em cada dois casos entre 20 e 39 anos19 .Por outro lado, as limitações do diagnóstico da infecção pelo HIV na população indígena masculina podem estar associadas à baixa cobertura de exames diagnósticos, ausência dos homens nas aldeias por motivos laborais e reduzida procura masculina pelos serviços de saúde. A baixa cobertura diagnóstica favorece a disseminação da infecção, pois o desconhecimento da doença leva à continuidade de práticas sexuais sem prevenção. A inclusão gradual dos testes rápidos para HIV nas aldeias a partir de 2012 pode ter contribuído para a retomada no crescimento da taxa de detecção de HIV nos últimos dois anos do período de estudo, uma vez que proporcionou o aumento da cobertura diagnóstica, como observado em outros DSEI do BrasilA observação das taxas de mortalidade e letalidade registradas entre os Kaiowá é relevante para a organização dos serviços de saúde, pois indica que essa etnia tem menor acesso ao tratamento antirretroviral. A diminuição das taxas de mortalidade e letalidade no Polo de Dourados, no segundo período, sugere maior estruturação dos serviços de saúde nas aldeias e articulação com os serviços de atenção especializada (SAE) para o tratamento dos indígenas com aids. O comportamento diferenciado das taxas de HIV e aids em cada um dos Polos mostra a necessidade de avaliação dos serviços de saúde separadamente, uma vez que eles se estruturam de forma diferenciada para enfrentar o problema.As principais limitações deste estudo estão relacionadas ao uso de fontes de dados secundários, que impõe entraves à qualidade da informação. A deficiência na completude da variável raça/cor indígena no Sinan e a subnotificação de casos de HIV e aids em indígenas são fatores que influenciam no número de casos identificados, e consequentemente, na cobertura da informação.g e em 2014 para a população em geralh . A subnotificação e incompletude dos dados da variável raça/cor dificultam a caracterização de situações de iniquidade em saúde e o estabelecimento de estratégias para o seu enfrentamento26 .Para ampliar a cobertura de casos de infecção pelo HIV, este estudo utilizou os registros de atendimentos diários das EMSI como fonte de dados, tendo em vista que a notificação compulsória foi incluída no Sinan somente em 2005 para gestantesA despeito destas limitações, o presente estudo trouxe benefícios por abordar uma doença particularmente pouco conhecida na epidemiologia dos povos indígenas no Brasil, que traz impactos no perfil de morbimortalidade, e por se relacionar à emergência de infecções sexualmente transmissíveis e sua associação com as mudanças socioculturais resultantes do processo de interação com a sociedade não indígena.27 e de articulação dos sistemas de saúde indígena e não indígena28 . O conhecimento das dificuldades logísticas, distribuição da população e áreas mais vulneráveis ajuda a definir estratégias para aumentar a cobertura diagnóstica e facilitar o acesso ao tratamento29 . Nesse sentido, a capacitação dos recursos humanos para trabalhar em um contexto intercultural é fundamental para aumentar a capacidade de resposta do Sasi-SUS nas ações de prevenção e tratamento das doenças.O desafio de trabalhar as IST em um contexto interétnico implica a necessidade de apreensão de conhecimentos sobre sexualidade inerentes a cada grupo indígenaO estudo evidenciou que o caráter endêmico do HIV e da aids, com predomínio dos casos de infecção pelo HIV na região sul do estado, pode se tornar epidêmico em alguns anos, pois já há casos em outras áreas indígenas do Mato Grosso do Sul. Entre as medidas de intervenção importantes para enfrentar o problema, destacam-se o aumento da cobertura diagnóstica, a garantia do acesso ao tratamento e as medidas de prevenção da transmissão do HIV.


The desired dual-functionalized POSS fillers were prepared, and because of its high miscibility, homogeneous films were readily obtained, by the casting method, with the mixture solutions containing the modified POSS and the polymers. From optical measurements, it was found that the larger effects of lowering the RIs of the PMMA matrices were observed from the modified POSS than those of the octa-substituted POSS derivatives with the homogeneous substituents. It should be mentioned that the degradation temperatures and the storage moduli were able to be greatly elevated by loading the present POSS fillers. Finally, it was demonstrated that the methacrylate ester-tethered POSS should be the most effective filler for modulating PMMA (Δ Organic−inorganic polymer hybrids , where bTuning of material properties of low-RI polymers has attracted tremendous attention in the development of modern electronic devices. As an anti-reflection film, low-RI polymers can contribute to the improvement of display resolution by suppressing light scattering, as well as enhancement of device efficiency, by facilitating light extraction . PerfluoWe have previously reported the design strategy for a molecular filler which can simultaneously lower RIs and enhance the durability of conventional polymers, by employing POSS that have dual types of functional groups ,35. The Herein, we demonstrate molecular fillers by employing POSS as a platform for modulating the optical and thermomechanical properties of the PMMA. Series of the dual-functionalized POSS derivatives that had seven fluorinated alkanes and a single functional group, such as methyl methacrylate ester, cyclopentyl, and octadecyl, on the silica cube, were prepared and loaded onto the PMMA film. Initially, it was confirmed that the present POSS fillers had higher miscibility than the octa-substituted POSS, with the homogeneous substituents. In addition, the larger filler effects on the optical and thermomechanical properties were obtained. The mechanism is discussed in this manuscript.1H, 100 MHz for 13C, and 80 MHz for 29Si) spectrometer. Coupling constants  are reported in Hertz. MASS spectra were obtained on a Thermo Fisher Scientific EXACTIVE spectrometer, for atmospheric pressure chemical ionization (APCI). Scanning electron microscopy (SEM) images were obtained using a JEOL JSM-5600 operator with an accelerating voltage of 10 kV. UV-vis transmittance spectra were recorded with a SHIMADZU UV-3600 UV-vis-NIR spectrophotometer. The refractive indices were determined with an Abbe refractometer DR-M4  at 580 nm at 25 °C. Dynamic mechanical analysis (DMA) was performed on a SDM5600/DMS210 , Tokyo, Japan) with a heating rate of 2 °C/min at 1 Hz, with 1% strain, under air. Differential scanning calorimetry (DSC) thermograms were carried out on an SII DSC 6220 instrument, SII, by using ~10 mg of exactly weighed samples, at a heating rate of 10 °C/min, under nitrogen. The glass transition temperatures (Tg) were evaluated from the second monitoring curves, after annealing at 100 °C for 10 min, followed by cooling to 30 °C. Thermogravimetric analysis (TGA) was performed on an EXSTAR TG/DTA 6220, SII, with a heating rate of 10 °C/min up to 500 °C, under nitrogen atmosphere. Residual chloroform was removed by keeping it in a vacuum oven, at 100 °C for 1 h before the TGA measurements. The van der Waals volumes of the POSS derivatives and the monomer unit of the PMMA were calculated after the modeling, using a semi-empirical AM1 method.NMR spectra were measured with a JEOL EX–400  was purchased from the Nacalai Tesque . Tetrahydrofuran (THF) and triethylamine were purchased from FUJIFILM Wako Pure Chemical Corporation and Kanto Chemical Co., Inc., Tokyo, Japan and were purified using a two-column solid-state purification system .Trimethoxysilane , 3-(trichlorosilyl)propyl methacrylate , trichlorocyclopentylsilane (Adrich), trichloro(octadecyl)silane (Adrich), and sodium hydroxide  were purchased and used for the assays, without further purification. PMMA (3O12Si7(C3H4F3)7]. Na3O12Si7(C3H4F3)7 was synthesized by the following method. Trimethoxysilane , THF (25 mL), deionized water , and sodium hydroxide  were charged in a round-bottomed flask, equipped with a reflux condenser. After being refluxed for 5 h, the mixture was cooled down to the room temperature and held, with vigorous stirring, for 15 h. The solvent was removed by a rotary evaporator and a white solid was obtained. After being dried in vacuo, 3.6 g of the products were obtained with a 97% yield.Synthesis of the Silsesquioxane Partial Cage  calcd. 1256.9853, found 1256.9864.F+MMA POSS: 1H NMR  δ 2.14 , 1.80 , 1.69−1.37 , 1.14−0.80 ; 13C NMR  δ 127.1 , 27.7 , 27.2, 26.9, 21.5, 4.0; 29Si NMR  δ –65.7, –67.5, −67.7; HRMS (APCI) [(M + Cl)−] calcd. 1198.9798, found 1198.9794.F+CP POSS: 1H NMR  δ 2.11 , 1.57 , 0.89 , 0.63 ; 13C NMR  δ 127.2 , 32.9, 32.1, 29.9, 29.5, 27.9, 22.8, 14.1, 4.11; 29Si NMR  δ –65.3, –67.5, −67.8; HRMS (APCI) [(M+Cl)−] calcd. 1383.1989, found 1383.2002.F+C18 POSS: Preparation of the Polymer Composites. The mixtures (20 mL) containing 1 g of PMMA and various amounts of POSS fillers in chloroform, were stirred at room temperature for 3 h and then poured into the bottom of the vessels (7 cm × 4.5 cm). After drying at room temperature for 2 days, the film samples were dried again, in a vacuum oven, at 60 °C for 2 h. The resulting films were used for the following measurements.α), molar refractions (R), and the molar volumes (V) [Determination of the Refractive Indices of the Polymer Composites. According to the Lorentz–Lorenz equation, the refractive index (n) of the polymer (element 1) composites containing fillers (element 2) can be described using the molar fractions (umes (V) ,42.Kp that is defined asVint and VVDE are the intrinsic and van der Waals volumes of the molecules, respectively. Therefore, molecules that have significant abilities to lower the interactions between polymer chains would show a smaller Kp value. In addition, according to the Vuks equation, Equation (1) can be transformed in the Equation (3).The degree of packing can be described by the molecular packing coefficient Kp values (Kp1) were determined to be 0.677 [Kp2 values by measuring the refractive indices of the composite (n), using Equation (3).In the case of amorphous polymers, the be 0.677 . TherefoTo meet opposite demands, the modified POSS derivatives that had the fluoroalkyl and other functional groups, were designed . AccordiThe PMMA films containing 2 mol% (ca. 20 wt%) F+MMA POSS and F+CP POSS mixtures had good transparency, whereas white opaque films were obtained from the F+C18 POSS . From thn = ca. −0.015). It should be mentioned that degree of the lowering effect of the F+CP POSS was much larger than that of the previous dual-functionalized POSS with cyclopentyl groups (ca. −0.005). These data clearly indicated that the fluorinated POSS can have a superior ability to be an efficient filler, for lowering the refractive indices of the PMMA. It is suggested that the POSS mixtures could be completely dispersed in the polymer matrices, by suppressing the crystallinity of the POSS, as mentioned above. Therefore, a lower density region should be efficiently created around the POSS filler, leading to the lowering effects on the refractive indices. According to previous reports, the degree of decrease in RIs, by the present POSS fillers, is large enough for applications in optical fibers [To evaluate the influence of the POSS fillers on the RI values of the PMMA, optical measurements were performed with the film samples containing variable concentrations of the POSS fillers, using an Abbe refractometer . The avel fibers ,44. ThusKp1) of the pristine PMMA film was determined as 0.677 [Kp2 value than the Kp1 would be obtained  Td20, using the polymer composites containing the POSS fillers. From the curves, it was indicated that degradation occurred through three steps. It was reported that pyrolysis of PMMA proceeds, initially, at the head-to-head linkage, followed by the chain-ends, and the random scission in the main-chains [Td20 values of the polymer matrices [Td20 at 215 °C, and significant thermal reinforcement of the PMMA matrices by loading the POSS fillers can be observed. By increasing the amount of the POSS, the Td20 values increased. Finally, the Td20 value of the PMMA matrices containing 2 mol% of the F+MMA POSS and the F+CP POSS increased by 53 and 11 °C, respectively. Comparing to the previous dual-functionalized POSS (ΔTd20 = +2 °C), larger stabilization effects were obtained. According to the previous reports, the methacrylate ester group connected to the POSS reacted with the PMMA matrices, by heating around 60 °C [Thermal stabilities of the polymer films containing fluorinated POSS fillers were examined by the TGA. n-chains . Accordimatrices ,29,34,40Tg values were slightly influenced by loading the POSS onto PMMA, with high homogeneity. Meanwhile, significant decreases in Tgs were observed in the heterogeneous films , although each POSS molecule created a lower density region. The E’ value was enhanced by 72% with the F+PMA POSS and 71% with the F+CP POSS. In the previous reports, loss of E’ was induced with the POSS filler possessing the hepta-substituted cyclopentyl and single fluoroalkyl groups [Finally, the mechanical properties of the polymer matrices containing the POSS fillers were evaluated by DMA (temperature scan at 1 Hz). The mechanical properties of the polymer composites containing the POSS fillers are listed in l groups . MoleculBy utilizing the molecular fillers based on fluoroalkyl POSS having dual types of functional groups, the polymer hybrids were prepared. From the series of measurements with the hybrid films, it was clearly shown that the POSS fillers can effectively lower the RI values of the PMMA and enhance both the thermal and the mechanical durability. It is proposed that the one functional group should make a strong interaction with the polymer chains, even in the presence of low-miscible hepta-substituents of the fluoroalkyl groups, followed by suppressing the molecular motions. These results indicate that the dual types of functional groups on the POSS core can share roles in simultaneously improving the thermomechanical properties and lowering the refractive indices of the conventional polymers. In other words, it was demonstrated that the POSS played a role only in introducing the positive effects of each functional group, followed by reconciling the trade-off relationship in the polymer functions. Furthermore, it can be said that our concept of designable hybrids, utilizing the preprogrammed-modified POSS could include a huge potential for receiving multi-functional polymeric materials with high stability.


Rising antibiotic resistance poses a challenge to the management of febrile neutropenia in patients with haematological malignancies receiving chemotherapy.We studied an alternating first-line antibiotic strategy to determine its impact on all-cause mortality and bacteremia rates in patients with febrile neutropenia.An alternating first-line antibiotic strategy was established in mid-2013. Data for 2012 (before strategy implementation) and 2014 (post-strategy implementation) were compared. Antibiotic Heterogeneity Index (AHI) for each of the two time-periods was also calculated.Staphylococcus aureus (MRSA) were 2.38/1000 and 2.59/1000 patient-days in 2012 and 2014 respectively. Rates of new cases of Vancomycin-resistant Enterococcus (VRE) were 1.84/1000 and 1.81/1000 patient-days in 2012 and 2014 respectively. There was no Carbapenem-resistant Enterobacteriaceae (CRE) bacteremia in 2012 and 1 in 2014.There were 2012 admissions (26082 patient-days) in 2012 and 1843 admissions (24331 patient-days) in 2014. There was no significant difference in the baseline characteristics of patients in the two groups. The defined daily doses (DDD) of cefepime (CEF) fell while the DDD of piperacillin-tazobactam (PTZ) rose in 2014 compared with 2012. Vancomycin DDD fell in 2014. The AHI was 0.466 in 2012 and 0.582 in 2014. The difference in all-cause mortality was not statistically significant. There was no difference in rates of bacteremia with CEF-resistant, PTZ-resistant and carbapenem-resistant gram-negative organisms in the two groups. Rates of new cases of Methicillin-resistant An alternating first-line antibiotic strategy resulted in an increase in antibiotic heterogeneity, without increasing mortality. There was also no significant increase in bacteremia rates. Empirical antibiotics are crucial in the management of fever in patients who are neutropenic after chemotherapy for haematological malignancies. Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species) which are the leading cause of nosocomial infections among immunocompromised individuals and are often characterized by antibiotic resistance. [Rising antibiotic resistance, however, threatens to limit the options for empirical and definitive treatment. Infections with multi-drug resistant organisms (MDROs) carry a high mortality, especially in the haematology-oncology setting. –4 Of paristance. Judicious antibiotic use, generally through an antibiotic stewardship program, is recommended as part of a multipronged approach against MDROs.  Like othAntibiotic cycling has been mooted as one way in which the development of resistance may be prevented.  The prinUnfortunately, the results of cycling studies have been mixed. In a bone marrow transplant unit, cycling was associated with stable rates in gram-negative resistance but a rise in ampicillin and vancomycin resistance among Enterococci.  In anothet al in an ICU. [Acinetobacter baumannii and extended spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae were high, but fell during the more heterogeneous period. [et al again showed that during the “mixing” period, there was a lower rate of resistant ESKAPE organisms. [In addition to antibiotic cycling, mathematical models have shown that heterogeneous antibiotic use  could reduce the emergence of resistance. A strate an ICU.  During p period.  In a repganisms.  A Japaneganisms. Our hospital is a 1700 bed tertiary hospital. In the Department of Haematology (DoH), cefepime (CEF) had been the first-line empirical antibiotic in febrile neutropenia for several years. [We hypothesized that antibiotic heterogeneity might retard the inexorable march towards pan-drug resistance, without significantly affecting mortality.In view of data negative for cycling and promising for antibiotic heterogeneity, increasing antibiotic heterogeneity was considered a viable approach to the rising problem of MDROs. After a consensus meeting comprising haematologists and infectious diseases physicians, it was decided that the choice of empiric (i.e. first-line) antibiotic for febrile neutropenia would be piperacillin-tazobactam (PTZ) on odd dates, and a cefepime-amikacin (CEF-AMK) combination on even dates. See  PatientsWe embarked on this before-and-after study to assess the utility and consequences of this use of alternating first-line antibiotics in febrile neutropenia. The primary objective was to determine if there was any impact on all-cause mortality in 2014 (after strategy implementation) compared to 2012 (before strategy implementation). The secondary objectives were to determine differences in rates of the following between the two study periods: bacteraemia-associated deaths, new cases of MRSA, new cases of VRE, CEF-resistant Gram-negative bacteraemia, PTZ-resistant Gram-negative bacteraemia and carbapenem-resistant Gram-negative bacteraemia. “New cases” of MRSA and VRE included those infected with these organisms, as well those who were colonized.et al. [We collected data on baseline characteristics of patients, disease-type and clinical outcome for the years periods 1 January 2012–31 December 2012 and 1 January 2014–31 December 2014, for two Haematology wards . In addition, we also collected the following data for these two periods: bacteraemia (including rates of bacteraemia caused by carbapenem-resistant organisms), new cases of MRSA, new cases of VRE and mortality. We calculated the incidence density of carbapenem-resistant Enterobacteriaceae (CRE) in patients admitted to the DoH and compared it with the rest of the hospital. All microbiology data were obtained from the hospital’s Laboratory Information System, and individually reviewed by the investigators. The defined daily doses (DDD) of CEF, PTZ, IMI and MER for 2012 and 2014 were calculated. All drug use data were obtained from the Pharmacy. The Antibiotic Heterogeneity Index (AHI) was calculated according to the formula used by Sandiumenge et al.  This stuIn the DoH, ciprofloxacin prophylaxis is provided routinely for all patients expected to experience prolonged neutropenia. Blood cultures will be drawn, and antibiotics may be commenced or adjusted when a neutropenic patient spikes a fever. Blood cultures drawn from each patient consist of two aerobic and two anaerobic bottles (BACTEC Plus Aerobic/F and BACTEC Plus Anaerobic/F respectively), as well as one dedicated fungal bottle (BACTEC Myco/F lytic), from at least two different sites. These were not changed in the two periods of the study.All new admissions are screened for MRSA  and VRE . From August 2014, DoH admissions were also screened for CRE via rectal swabs.C. difficile diarrhoea (for up to two days after diarrhoea stops). Patients who test positive for a respiratory virus are isolated in single rooms. These were not changed in the two periods of the study.Due to a limited number of single rooms, single rooms are only used to house the following categories of patients: MRSA respiratory dispersers, VRE-positive patients with diarrhoea and patients with Please refer to 3 or an ANC that was expected to decrease to <500 cells/mm3 during the next 48 hours. [Staphylococcus, Bacillus, Corynebacterium or other skin contaminants, for which growth from at least two bottles, taken at different times, was needed. New cases of MRSA or VRE was defined as the isolation of that organism from either clinical or screening specimens in a patient who had never been positive previously, if the isolate grew from a sample submitted ≥48 hours after hospital admission. For multiply-admitted patients, a positive admission screen made the patient a new case, if previous admission screens were negative.Fever was defined as a single temperature measurement of >38.3°C 101°F) or a temperature of >38.0°C (100.4°F) sustained over a 1-hour period.[01°F or aBaseline characteristics of the two groups were summarized using descriptive statistics and the chi-square test was used to compare non-continuous variables. Antibiotic usage was expressed as DDD (defined daily doses) per 1000 patient days. The Poisson distribution method was used to calculate the 95% confidence interval (CI) for the incidence rate ratio of bacteraemia, new cases of MRSA and VRE between the two groups. A p-value of <0.05 was considered statistically significant.In 2012, there were 2012 admissions (26082 patient days) while in 2014 there were 1843 admissions (24331 patient days) to the two wards under study. The baseline characteristics of the patients admitted in these two different years are shown in Although there were more new diagnoses of acute myeloid leukaemia (AML) in 2012, the number of patients on chemotherapy for AML in 2012 was not different statistically from 2014. There was no difference in the number of admissions for the other malignant conditions, e.g. myeloma.  The spreIn 2014, the DDD of CEF fell , and the DDD of PTZ rose .  The DDD The rate of use of other antibiotics was not different statistically. The AHI was 0.466 in 2012 and 0.582 in 2014. There was no change in the rates of new MRSA and new VRE cases in the years 2012 & 2014.  The diffet al when one half of treated patients are assigned to one drug, and the other half to another. (11) Bergstrom et al show that the proportion of patients colonized with resistant organisms is greater for any cycling interval compared with 50–50 mixing. (11) Our day-by-day alternation of empirical first-line antibiotics is the closest strategy to the 50–50 mixing envisaged by Bergstrom et al.In this report, we demonstrate the clinical effects of subjecting a population of patients to a strategy of “mixing”. This is the term used by Bergstrom Most important, the strategy was safe. The primary outcome, all-cause mortality, was unchanged in the two observation periods. We could not run this as a randomized controlled trial, which is the major limitation. Non-consenting patients would mean that the strategy could not be assessed, as the strategy of alternating antibiotics targeted the ecology of the ward. We were unsure if the strategy would be followed in the wards. Mixed results from other studies were attributed, in part, to poor compliance.  Hence, tet al were restricted to an ICU setting. [Acinetobacter baumannii fell during the mixing period. [At least two other groups have attempted to introduce antibiotic heterogeneity with encouraging outcomes. The studies of Sandiumenge setting. , 15 They period.  In the set al adopted a different approach to try to achieve heterogeneity. [P. aeruginosa fell during the intervention period.Takesue geneity.  Their stet al or Sandiumenge et al. It is likely that the range of antibiotics used in the Haematology ward is not as broad as in other wards. Many patients are on a quinolone as it is our policy to use quinolones for febrile neutropenia prophylaxis. Amoxicillin-clavulanate and ceftriaxone are hardly used since, with febrile neutropenia in inpatients, only anti-pseudomonal antibiotics are recommended. [To our knowledge, our study represents the first attempt to bring about antibiotic heterogeneity in a Haematology setting. The AHI did rise after the alternate-day strategy was introduced, but not to levels achieved by Takesue mmended. The lack of a statistically significant difference in mortality and bacteraemia rates in the two years under study suggests that the mixing strategy was safe. Tables  & 5 StriWe were not able to show a difference in the rates of new cases of VRE or MRSA. Of the preventive measures against MRSA, the effective ones include improved hand hygiene, active surveillance followed by decolonization, and single-room isolation. As singlHowever, the CRE rate appeared not to have increased in our unit, despite a big increase hospital-wide. Papers describing attempts at CRE control have reported bundled interventions, making it difficult to tease out the component that worked. However In summary, alternating first-line antibiotics in febrile neutropenia in patients with hematologic malignancies resulted in an increase in antibiotic heterogeneity, without increasing mortality. There was also no significant increase in bacteraemia rates and compliance with the use of the strategy was excellent.S1 Appendix(DOCX)Click here for additional data file.


Fresh concrete used in 3D printing should ensure adequate yield stress, otherwise the printed concrete layer may suffer intolerable deformation or collapse during the printing process. In response to this issue, an analytical study was carried out to derive the initial yield stress and hardening coefficient of fresh concrete suitable for 3D printing. The maximum shear stress distribution of fresh concrete was calculated using a stress transformation equation derived from the equilibrium condition of forces. In addition, the elapsed time experienced by fresh concrete during the printing processes was estimated and was then substituted into the elapsed time-yield stress function to calculate the yield stress distribution. Based on these results, an algorithm capable of deriving both the initial yield stress and the hardening coefficient required for printing fresh concrete up to the target height was proposed and computational fluid dynamics (CFD) analyses were performed to verify the accuracy of the proposed model. Reinforced concrete (RC) structures have been widely utilized because they are extremely easy and economical to build. However, the disadvantages of the construction method include the facts that it causes an inefficient consumption of materials, problems with the labour force and issues with the construction period in the process of manufacturing the formwork, processing/placing the reinforcement and pouring the concrete . FurtherKhoshnevis  developeThe constitutive equations of fresh concrete are commonly represented by the Bingham model or the Herschel-Bulkley model . In the The purpose of this study is to quickly obtain the initial yield stress and hardening coefficient required to prevent the deformation and collapse of fresh concrete during printing. To this end, the maximum shear stress distribution generated in fresh concrete during the built-up process was calculated using the force equilibrium condition based on the slump prediction model of Murata  and the The yield stress of fresh concrete increases with the increasing elapsed time and thus the time at which fresh concrete is supplied affects buildability. The accelerator is injected from the nozzle, as shown in The cross section of fresh concrete is a perfect rectangle, as shown in Fresh concrete is built up vertically and the width  generated from any height z to an xy plane can be calculated as shown below [ρ is the density of fresh concrete and g is the gravity acceleration. The maximum shear stress (τmax ) that occurs in the layered fresh concrete can be calculated from the equilibrium condition of forces, as shown below [In wn below ,16,17,18wn below ,16,17,18xy plane can be calculated as shown below.In addition to the self-weight of fresh concrete, considering the extra shear stress  are a function with respect to If Equation  is substituted into Equations (21a), (21b), (22a) and (22b), the yield stress That is, the yield stress Deformation occurs when the maximum shear stress generated in layered fresh concrete exceeds the yield stress ,16,17,18Therefore, the yield stress  and (27b), the to Petit , the harComputational fluid dynamics (CFD) is one of the analytical techniques used to predict the behaviour of fluids. It has been widely used to simulate the behaviour of fresh concrete analysis . In thisβ), due to the injection of accelerator. The β of Case 2 is the hardening coefficient, which is calculated through the algorithm proposed in this study required to prevent the collapse of fresh concrete. In Case 1, the proposed model well predicted the CFD simulation results, in which the fresh concrete collapsed at a height of approximately 0.018 m. On the other hand, in Case 2, the fresh concrete did not collapse in both results by the proposed model and the CFD analysis.The proposed model could reflect the increase in the yield stress of the fresh concrete during the 3D concrete printing process by substituting the elapsed time into the yield stress-elapsed time equation.Based on the CFD analysis results on the cases examined in this study, it was confirmed that the proposed model can provide very accurate estimations on the occurrence and location of collapse.The proposed model would be very helpful to obtain the rheological properties of fresh concrete required for 3D printing, such as hardening coefficient and initial yield stress, without any complex numerical simulations.Further researches are still required to check the stability problems of the layered concrete for vertically complicated structures.This study aimed to develop an algorithm to quickly derive the rheological properties needed to prevent the collapse of fresh concrete during the printing process. In the proposed model, the fresh concrete was regarded as Herschel-Bulkley fluid and it was thus assumed that no deformation occurs before the maximum shear stress of layered concrete exceeds its yield stress. The maximum shear stress in the layered fresh concrete was calculated from the force equilibrium condition and the yield stress of the fresh concrete at any location during the printing process was estimated considering the elapsed time that depends on the printing path. Based on this research, the following conclusions were drawn:


Few data are available about temporal trends of antibiotic use in hospitalized children. The aim of the current study was to investigate the pattern and trends of antibiotic use over the years 2008–2016 in the largest children’s hospital in Italy.Annual point prevalence surveys of antibiotic use were conducted by reviewing medical charts of 0–17 year-old children hospitalized for ≥48 h. Prevalence of antibiotic use was computed by year, type of ward and indication. Trends in prevalence over time were evaluated using the Cochrane-Armitage test. Possibile determinants of antibiotic use were assessed at univariate analysis and through a logistic regression model.p = 0.001). The prevalence of patients receiving antibiotics for medical prophylaxis increased from 6.1% in 2008 to 24.2% in 2016 (p < 0.001), whereas the prevalence of patients receiving antibiotics for surgical prophylaxis significantly decreased ; no significant temporal trends were found in antibiotic use for treating infections.Out of 3015 children, 1516 (50.3%) received antibiotics, 58.1% of which for medical/surgical prophylaxis. Prevalence of antibiotic use increased from 42.0% in 2008 to 56.2% in 2016 (p < 0.001), age , length of stay (LOS) , and type of ward  were significantly and independently associated with antibiotic use.The administration of third-generation cephalosporins for surgical and medical prophylaxis significantly decreased over time, while the proportion of antibiotics prescribed to treat infections after microbiological investigations significantly increased. Year  contains supplementary material, which is available to authorized users. Antibiotics are among the drugs most commonly prescribed to children, and are often used to treat common conditions generally caused by viral agents, against which antibiotics are mostly ineffective . In outpA judicious use of antibiotics is essential to slow the emergence of antibiotic resistance in bacteria, and to extend the useful lifetime of effective antibiotics . IncreasThere is a wide variability in antibiotic consumption between countries, and Italy is one of the european nations with the highest use of antibiotics, both in outpatient and inpatient settings . In 2007Few data are available about temporal trends of antibiotic use in hospitalized children . The aimThis study was conducted at the Bambino Gesù Children’s Hospital , a 607-bed tertiary care academic hospital in the Lazio Region, Italy. Due to a shift towards day-hospital and day-surgery care, the annual hospital inpatient admissions decreased from 33,132 in 2008 to 26,947 in 2016. Over this period, the average diagnosis-related group (DRG) weight - a measure of disease severity - increased from 0.86 in 2008 to 1.04 in 2016. The average length of stay (LOS) increased from 5.4 in 2008, to 6.5 in 2016. Starting 2008, a series of actions have been undertaken to promote the appropriate use of antibiotics, namely: a) dissemination of results of annual prevalence surveys on antibiotic use by posting reports on the hospital intranet website, presenting data in hospital meetings, and discussing actions to be undertaken within the hospital infection control team (from 2008); b) production and dissemination of hospital guidelines on antibiotic surgical prophylaxis (from 2009), c) production and dissemination of hospital guidelines on antibiotic medical prophylaxis (from 2011); d) restriction of use of third-generation cephalosporins for surgical prophylaxis (from 2012).We have conducted annual point prevalence surveys of hospital antibiotic use in years 2008–2016. The surveys were carried out during a maximum of two calendar weeks in the summer period and involved all patients aged 0–17 years hospitalized for at least 48 h. Data were collected from patients’ paper medical charts. Information collected for each patient included: age, sex, length of hospital stay, and ward type. In case of antibiotic prescriptions, we collected information on antibiotic type according to the Anatomical Therapeutic Chemical Classification code, ATC J01) , drug brPatients were characterized according to demographic factors (age and sex), length of stay and ward of hospitalization. Wards were categorized as medical , surgical , medical subspecialty , and intensive care units .Prevalence of antibiotic use was the main measure of outcome. It was calculated as a ratio between the number of patients under treatment with at least one antibiotic on the day of the survey, and the total amount of patients hospitalized for at least 48 h. Prevalence of antibiotic use was computed by year, type of ward and indication. Antibiotic classes were stratified by indication of antibiotic prescription.Trends in patient characteristics and in prevalence of antibiotic use over time were evaluated using the Cochrane-Armitage test. Year, sex, age, length of stay and ward of hospitalization were investigated as possible determinants of antibiotic use. We evaluated the association between all these variables and exposure to antibiotics at univariate analysis and through a logistic regression model. Crude and adjusted odds ratios (ORs), with 95% confidence intervals (CI) were estimated.All statistical analyses were conducted using STATA 13 .p = 0.02); the proportion of inpatients in surgical wards decreased from 35.9 to 20.2% (p = 0.0001) whereas those hospitalized in Medical subspecialty unit increased from 21.1% in 2008 to 29.6% in 2016 (p = 0.01).A total of 3015 pediatric inpatients were involved in point prevalence surveys conducted between 2008 and 2016. The demographic characteristics of patients remained quite stable over the years (Table n = 1516). Among the hospital wards, intensive care units had the highest prevalence of antibiotic use, namely 62.0% (194/313 patients admitted in ICUs), followed by 52.1% in medical subspecialty units (449/861), 50.6% in surgical units (410/810), and 44.9% in medical units .The overall nine-year prevalence of antibiotic use was 50.3% (n = 515) and 24.1% (n = 366) respectively of all children receiving antibiotics. The proportion of patients who received antibiotics for treating CAIs or HAIs was equal to 24.9% (n = 378) and 21.5% (n = 326), respectively.Prophylaxis was the main indication for antibiotic use. Children receiving antibiotic for medical or surgical prophylaxis accounted for 34.0% ; no significant trends were found in antibiotic use for treating infections.Trend analysis showed an increase in the prevalence of antibiotic use, from 42.0% in 2008 to 56.2% in 2016 (p < 0.001). Oncological patients accounted for 31.2% of patients treated with antibiotics in medical subspecialty units; this percentage increased from 18.8% in 2008 to 38.8% in 2016 (p < 0.001).When analyzing the prevalence of antibiotic use by indication and type of ward, we found that the rate of patients in surgical units who received antibiotic for surgical prophylaxis remained stable over time (from 31.6% in 2008 to 35.8% in 2016), while the rate of patients in medical subspecialty units who received antibiotic for medical prophylaxis significantly increased, from 13.8% in 2008 to 46.9% in 2016 (p < 0.01), age , and length of stay .The year was significantly and independently associated with antibiotic use Table . Other iThroughout the study period, third-generation cephalosporins ranked first both for the treatment of CAIs (where they were administered to 38.6% of patients), and for surgical prophylaxis  . The decrease in the administration of third-generation cephalosporins concerned both surgical  and medical prophylaxis .The use of combinations of penicillins, incl. β-lactamase inhibitors and combinations of sulfonamides and trimethoprim increased mainly in medical prophylaxis  and an increase in penicillins with extended spectrum  were found in patients treated for CAIs. Concerning HAIs, increasing trends in the use of imidazole derivatives and tetracyclines were observed .A reduction in glicopeptyde use . The proportion of treatments prescribed after microbiological testing was significantly higher for antibiotics prescribed for HAIs , compared to CAIs .Overall, a microbiological test was conducted in 636 (61.2%) treatments out of 1039 prescriptions for infections. This proportion went from 42.3% in 2008 (52/123) to 59.6% in 2016  and combinations of penicillins, incl. β-lactamase inhibitors resulted to be the antibiotic classes mainly used for medical prophylaxis, being widely used to prevent opportunistic infections in neutropenic patients . HoweverThe rate of inpatient children in surgical wards, who received surgical antibiotic prophylaxis remained stable over the years, however there was a significant improvement in the quality of molecules used. In fact, third-generation cephalosporins were the first-choice for surgical prophylaxis until 2012, when they were replaced by first- or second-generation cephalosporins, as recommended , 25. ThiFor hospitalized patients who have suspected infections, international guidelines reccomend to collect microbiological samples before prescribing an antimicrobial . PrematuPoint prevalence surveys have been used to assess antibiotic use in children , 29, 30,The results of this study should be interpreted in the light of some limitations. It was conducted in the largest tertiary care children’s hospital in Italy, where a series of actions were implemented to promote over the years a judicious use of antibiotics. Thus, we cannot assume that its results are representative of the in-hospital trend of antibiotic use in other centers. Information on the beginning of antibiotic therapy was not collected, so we cannot exclude the possibility that some children with CAIs were already under therapy when admitted, which limits the possibility of targeting the drug choice on microbiological results.Comparing prevalence rates of antibiotic use among hospitals and over time should consider differences in patient characteristics, such as age, ward of hospitalization and length of hospitalization. We documented an improvement in the choice of antibiotics prescribed for medical and surgical prophylaxis. However, further efforts are needed to avoid antibiotic misuse for medical prophylaxis, and to reduce the empirical use of broad spectrum antibiotics.Additional file 1: Trend analysis of the proportion of patients treated with antibiotics by indication and type of molecules . (DOCX 36 kb)


Parkinson’s disease (PD) is characterised by dopaminergic cell loss within the substantia nigra pars compacta (SNc) that leads to reduced striatal dopamine content and resulting motor deficits. Identifying new strategies to protect these cells from degeneration and retain striatal dopaminergic innervation is therefore of great importance. Chondroitin sulphate proteoglycans (CSPGs) are recognised contributors to the inhibitory extracellular milieu known to hinder tissue recovery following CNS damage. Digestion of these molecules by the bacterial lyase chondroitinase ABC (ChABC) has been shown to promote functional recovery in animal models of neurological injury. Although ChABC has been shown to promote sprouting of dopaminergic axons following transection of the nigrostriatal pathway, its ability to protect against nigrostriatal degeneration in a toxin-based module with better construct validity for PD has yet to be explored. Here we examined the neuroprotective efficacy of ChABC treatment in the full and partial 6-hydroxydopamine (6-OHDA) lesion mouse models of PD.In mice bearing a full 6-OHDA lesion, ChABC treatment failed to protect against the loss of either nigral cells or striatal terminals. In contrast, in mice bearing a partial 6-OHDA lesion, ChABC treatment significantly protected cells of the rostral SNc, which remained at more than double the numbers seen in vehicle-treated animals. In the partial lesion model, ChABC treatment also significantly preserved dopaminergic fibres of the rostral dorsal striatum which increased from 15.3 ± 3.5% of the intact hemisphere in saline-treated animals to 36.3 ± 6.5% in the ChABC-treated group. These protective effects of ChABC treatment were not accompanied by improvements in either the cylinder or amphetamine-induced rotations tests of motor function.ChABC treatment provided significant protection against a partial 6-OHDA lesion of the nigrostriatal tract although the degree of protection was not sufficient to improve motor outcomes. These results support further investigations into the benefits of ChABC treatment for providing neuroprotection in PD. Parkinson’s disease (PD) is typically characterised by a range of cardinal motor symptoms that result from dopaminergic cell loss within the substantia nigra pars compacta (SNc) . To dateTo date, the application of ChABC in the PD field has centred on demonstrating the ability of CS-GAG digestion to improve the success of dopaminergic cell replacement therapies –11, rathHere we examined the disease modifying potential of ChABC in the unilateral 6-hydroxydopamine (6-OHDA)-lesioned mouse which has good construct validity, replicating the mitochondrial dysfunction, oxidative stress and neuroinflammatory aspects of PD , 17. Accp = 0.46, t = 0.09, df = 25; Fig. F1,50 = 0.01, p = 0.89; Fig. F6,150 = 20.1, p < 0.001; Fig. F1,25 = 0.01, p = 0.91). Similarly, there was no effect of treatment, only time, on the degree of amphetamine-induced net ipsiversive rotations produced over the 90 min recording period  stub antigen that remains following ChABC-mediated digestion. C4S-immunoreactivity was detected along the entire nigrostriatal tract, including the SNc and striatum, of ChABC-treated animals Fig. g. Subseqp = 0.02, t = 2.4, df = 32; Fig. p = 0.48, t = 0.68, df = 32; Fig. p = 0.53, t = 0.6, df = 32; Fig. p = 0.007, t = 2.9, df = 32).ChABC treatment provided significant preservation of both SNc cells and striatal TH-positive fibres in the unilateral partial 6-OHDA lesion mouse model. Within the SNc, a significant difference in the percentage of cells remaining in the lesioned hemisphere was found between the saline- (24.8 ± 6.1% of intact hemisphere) and ChABC-treated animals (51.6 ± 8.5% of intact hemisphere) at the rostral level . Post-hoc analysis revealed a significant effect in dorsal striatum alone , it is clear that any resultant ChABC-mediated plasticity cannot counter the effects of a full lesion. This lack of efficacy is consistent with a previous study that showed no beneficial effect of ChABC treatment alone or in combination with DA grafts in a full 6-OHDA lesion model of PD . AlthougIn contrast, when administered acutely at the time of 6-OHDA infusion, ChABC did offer protection in the partial lesion model. Specifically, protection was afforded against the TH-positive cell loss in the rostral SNc, but not in either the medial or caudal levels of the SNc. Given that the pilot study revealed complete CS-GAG digestion throughout the extent of the nigrostriatal axis 35 days after ChABC injection, this regionally-restricted action of ChABC was unexpected. However, as one of the two ChABC injection sites was positioned near to the rostral SNc it is possible that these cell bodies were exposed to the enzyme more readily and to a higher degree than the rest of the SNc, providing these with optimal protection. Interestingly, the accompanying preservation of TH-positive fibres afforded by ChABC treatment was also restricted to the rostral striatum. Whether this is also a consequence of the location of ChABC injection or a reflection of the topography of dopaminergic projections from the SNc to striatum requires further investigation.The molecular mechanisms behind ChABC’s protective effect have not been explored here. However, digestion of the CS-GAGs is believed to liberate ligands such as various growth factors that aid plasticity –25. GiveDespite an increase in numbers of surviving dopaminergic neurones in the rostral nigrostriatal tract, ChABC treatment did not result in any behavioural improvement in either the amphetamine-induced rotation or cylinder test assessments. This lack of motor improvement may reflect the modest sparing afforded by ChABC treatment of both rostral SNc cells (reaching only 52% of the intact hemisphere cell number) and striatal TH fibres . Conversely this may reflect the fact that there was still a relatively large lesion size in the partial lesion model used here, with only 25% cells in the SNc and 36% striatal TH fibres remaining in the saline-treated animals bearing a partial lesion. Indeed, recent neuroprotective studies utilising the unilateral 6-OHDA mouse model which identified a significant improvement in behavioural phenotype following various treatments support the starting lesion size being the main obstacle to achieving a behavioural improvement. In these previous studies, the lesions were less severe to start with  and so, with an even more modest degree of protection than observed here , this was sufficient to elevate nigrostriatal tract functionality –34. It wA number of alternative avenues for improving the behavioural outcome with ChABC treatment deserve consideration. Firstly, as the ChABC enzyme is believed to become denatured quickly under physiological conditions, increasing the exposure to ChABC through varied means which have proven successful against spinal cord injury such as repeated intrathecal administration , gene thFinally, given that new neuronal connections are theorised to be of use only once the animal has learnt to use them through successful rehabilitation, future studies exploring the benefits of combining ChABC treatment with rehabilitation are warranted , 41. UtiWhile the 6-OHDA model utilised here is considerably better in terms of construct validity that the previously explored transection models –15, it iThis study has revealed the efficacy of ChABC treatment in the 6-OHDA partial lesion mouse model of PD, whereby digestion of CSPGs has reduced the degree of pathology within the nigrostriatal tract. Although it remains to be established whether combined interventions such as rehabilitation can improve motor outcomes, these findings present promise for ChABC treatment as a potential new avenue for providing neuroprotection in PD. In this respect, it is reassuring that alternative formulations with enhanced stability  and alte27 and 34 eight-week-old male C57Bl/6 mice  were used within the full lesion and partial lesion studies, respectively. All 61 mice were maintained on a 12:12 h light/dark cycle (07:00 am lights on) with food and water ad libitum. Room temperature and humidity were kept at 22 ± 2 °C and 55 ± 2% respectively. All in vivo studies were performed in accordance with the UK Animals Scientific Procedures Act (1986) and were approved by King’s College London Animal Welfare and Ethical Review Body. All surgical, behavioural and histological procedures were performed whilst blinded to the experimental groups.All surgeries were conducted in a randomised block design to reduce bias between the blinded treatment groups. Fully lesioned animals were assigned to ChABC treatment (n = 13) or saline control (n = 14). Partially lesioned animals were assigned to ChABC treatment (n = 17) or saline control (n = 17). Anaesthesia was induced with a 5% isoflurane/oxygen mixture then maintained at 3% isoflurane/oxygen. Body temperature was monitored and maintained at 37 °C with a homeothermic heating blanket (Harvard Apparatus). The surgical site was sterilised with 0.4% chlorhexidine (Hibiscrub) before a midline incision was made along the scalp. The skull was then cleaned and dried with cotton swabs.To establish a full unilateral 6-OHDA lesion, fine-bore holes (Ø 0.5 mm) were drilled at coordinates AP: − 3.0 mm and ML: + 1.2 mm (relative to bregma and skull surface). A blunt-ended 30G needle was then inserted supranigrally to DV: − 4.5 mm (relative to bregma and skull surface) before 8 μg 6-OHDA·HBr (Sigma-Aldrich) in 1 μl 0.02% ascorbate/saline was administered unilaterally at a rate of 0.5 μl/min. The needle was left in place for 4 min to allow for toxin diffusion.To establish a partial unilateral 6-OHDA lesion, fine-bore holes (Ø 0.5 mm) were drilled at coordinates AP: + 0.5 mm and ML: + 2.0 mm (relative to bregma and skull surface). A blunt-ended 30 G needle was then inserted intrastriatally to DV: − 3.5 mm (relative to bregma and skull surface) before 4 μg 6-OHDA·HBr in 1 μl 0.02% ascorbate/saline was administered unilaterally at a rate of 0.5 μl/min. The needle was left in place for 4-min to allow for toxin diffusion.5-min after injection of 6-OHDA, animals from both studies received two intracerebral injections of either saline or ChABC  into the same 6-OHDA injected hemisphere. In order to achieve digestion of CSPGs along the entire nigrostriatal tract, 1 μl ChABC was administered into the SNc  and 1 μl ChABC into the striatum . These coordinates were determined by a previously run pilot study that showed these to provide most effective CSPG digestion along the entire nigrostriatal tract (data not shown).After the surgery, all animals were administered buprenorphine  for analgesia. All animals additionally received 1 ml of warmed Hartmann’s solution  twice a day for 1 week to maintain hydration.Motor dysfunction was assessed using two behavioural tests. The cylinder test measured the degree of forelimb ability as described previously . Brieflyd-amphetamine hemisulphate  and rotations were recorded for 90-min thereafter.Amphetamine-induced rotations were counted on day 35 (full) or day 37  post-lesion. Animals were placed within cylindrical arenas (Ø 40 cm) in which the motion-tracking tool Ethovision XT6 was used for recording. A custom optimised calibration file was loaded, which allowed the recording of full 360° rotations about the animal’s midpoint. Ipsiversive and contraversive rotations were then individually measured before calculating net ipsiversive rotations. Following a 20-min habituation period, mice were administered with l-lysine coated slides (VWR). Three sections from each of the three levels of the striatum  were incubated with rabbit polyclonal anti-tyrosine hydroxylase (TH) primary antibody  overnight before being washed in TBS twice and incubated in biotinylated goat anti-rabbit secondary antibody  for 2 h at room temperature. Following a further two TBS washes, the sections were incubated with streptavidin-biotinylated horseradish peroxidase conjugate  for 30-min at room temperature. Slides were then immersed in diaminobenzidine tetrachloride for 10-min and mounted.On day 42 post-lesion (full lesion study) or day 39 post-lesion  all animals were humanely killed following sodium pentobarbital terminal anaesthesia . Animals were then formalin-perfused and their brains post-fixed in 4% paraformaldehyde/PBS before being embedded in paraffin wax blocks. 7 µm thick coronal sections were cut with a microtome (Thermo Scientific) at three rostrocaudal levels of the SNc  and three levels of the striatum . Sections were then mounted on Poly-Cells of the SNc and TH-positive fibres of the striatum, in both the lesioned and intact hemispheres, were imaged with 100× and 50× magnification, respectively . For the purpose of side-to-side comparisons where the intention is to quantify percentage loss between the intact and lesioned hemispheres in a given animal, we adopted manual cell counting . PreviouImmunohistochemistry was used to stain for the C4S-stub antigen that remains following ChABC-mediated digestion. Staining for C4S  was completed using sections adjacent to the TH-stained sections whilst using the same protocol as previously described for TH .To confirm whether the extent of ChABC-mediated digestion of CS-GAGs along the nigrostriatal pathway in vivo was maximal, the effect of subsequent ChABC exposure ex vivo was assessed. Briefly, 7 µm SNc brain sections from saline- and ChABC-treated mice from both the full and partial lesion studies (n = 3 for each group) were incubated with ChABC  or tris-buffered saline for 3 h at 37 °C. C4S-stub immunoreactivity was then stained for, as described above.t-tests and striatal TH-positive fibre MGV data were analysed by two-way ANOVA. Post-hoc tests were applied when appropriate as detailed in figure legends.All quantitative data are expressed as mean ± standard error of the mean (SEM). Statistical analyses were conducted with GraphPad Prism (version 7) software. Behavioural data were analysed using two-way repeated measures ANOVA, SNc cell count data and TH density were analysed by unpaired Student