Complications of care in hospitalization for type 2 diabetes mellitus: the situation of urinary infections

Provas públicas apresentadas à Escola Superior de Saúde Dr. Lopes Dias do Instituto Politécnico de Castelo Branco para o cumprimento dos requisitos necessários à obtenção do título de Especialista.Objetivo: O objetivo primário consistiu em estudar a prevalência da taxa das infeções urinárias durante o internamento; e o objetivo secundário: identificar os fatores explicativos para estas infeções. Métodos: Estudo transversal com recolha de dados retrospetivos no período de 01 janeiro a 31 de dezembro de 2015. Utilizou-se a base de dados dos resumos de alta para os episódios de internamento da Diabetes mellitus tipo 2 (DMTII), como diagnóstico principal. Na primeira abordagem usou-se a análise estatística descritiva bivariável onde se calculou a distribuição e a taxas das infeções urinárias. Na segunda abordagem usou-se a regressão logística com recurso ao modelo de ajustamento pelo risco: as infeções urinárias constituíram a variável dependente; o género, a idade, a gravidade da doença principal, as co morbilidades e respetivos níveis de gravidade, o número de dias de internamento, o hospital, o tipo de procedimento e os GDH, as variáveis independentes. Resultados: As taxas das Infeções Urinárias apresentaram um valor de 16,2%. Os diabéticos de gravidade (1) apresentaram uma taxa de 24,4% contra 4,8% nos diabéticos de gravidade (2). No tipo de tratamento médico a taxa foi de 24,6% contra 4,8% no tratamento cirúrgico. Os fatores de risco como a idade (OR=1,031), o número de dias de internamento (OR=1,018), o género feminino (OR=2,248), a gravidade (3) da co morbilidade Doença Cerebrovascular (NEU04) (OR=1,737), a gravidade (1) da co morbilidade Neoplasia maligna do cólon e reto (GIS27) (OR=2,417), bem como dez hospitais tiveram influência sobre a ocorrência de COC Urinárias. Conclusões: Estes resultados podem ser justificados por práticas clínicas distintas e pela adoção de protocolos de cuidados pouco seguros, pelo que é necessário implementar uma vigilância dirigida e um reforço de prevenção e controlo através da elaboração de modelos de segurança mais efetivos.Abstract Context: The primary objective was to study the prevalence of the rate of urinary infections during hospitalization; and the secondary objective: to identify the risk factors for these infections. Methodology: A cross-sectional study with retrospective data collection from January 1st to December 31st, 2015. The database of the discharge summaries for the hospitalization episodes of Type 2 Diabetes Mellitus (DMTII) was used as the principal diagnosis. In the first approach we used the bivariate descriptive statistical analysis where the distribution and rates of urinary infections were calculated. The second approach used the logistic regression using the risk-adjustment model: the COC Urinary was the dependent variable; sex, age, the severity of the principal disease, the severity of the comorbidities, number of hospitalization days, hospital, type of procedure and GDH, were independent variables. Results: The principal results found indicate that the COC Urinary rate was of 16.2%. Diabetics of stage (1) the rate was 24.4% versus 4.8% in diabetics of stage (2). In the type of medical treatment, the rate was 24.6% versus 4.8% in the surgical treatment. The risk factors, such as age (OR=1.031), number of hospitalization days (OR=1.018), female gender (OR=2.248), the stage (3) of comorbidity Cerebrovascular disease (NEU04) (OR=1.737) and the stage (1) of comorbidity Malignant neoplasm of colon and rectum (GIS27) (OR=2.417), as well as ten hospitals had influence over the occurrence of COC Urinary. Conclusion: These results can be justified by different clinical practices and the adoption of unsafe care protocols. These findings lend support to the importance of implementing a targeted surveillance and development effective models of patient safe in COC.N/

Robust design and operation of quasi-continuous adenovirus purification by two-column, simulated moving-bed, size-exclusion chromatography

Adenoviruses (Ads) are considered one of the most suitable platforms for production of viral vaccines and gene therapy vectors. Their broad tissue tropism and large transgene packing capacity make them attractive candidates for innovative virotherapies. Ads can be produced in a complementary cell line in both adherent and suspension culture systems, such as HEK-293 or PER-C6 cells, or A549 for oncolytic therapies. Liquid chromatography is currently the core technique for vector purification, and its use is often integrated vertically within the downstream processing (DSP) strategy, as it easily fits into the early capture stage as well as into the final purification phase. The use of high-performance liquid chromatography (HPLC) for large-scale adenoviral purification is an already established method, including ion exchange (IEX), size exclusion (SEC), hydrophobic interaction, and immobilized metal affinity chromatography. Unlike traditional processes based on CsCl-gradient purification, HPLC offers a straightforward linear scale-up path, and procedures for purifying up to around 1014 input particles have been reported. SEC and ultra/diafiltration (usually by tangential flow filtration) are two other widely used processes at the very latest stage for formulating the product. One process-based way to improve the performance of the DSP chromatographic steps is by changing to a (quasi-)continuous processing mode, which, in principle, yields higher throughput, lower buffer consumption, higher capacity utilization of the stationary phase and reduced column volume, hence increased productivity. In this work, a simple, yet efficient, two-column simulated moving-bed (2CSMB) process for purifying adenovirus serotype 5 (Ad5) by size-exclusion chromatography (SEC) is presented and validated experimentally, and a general procedure for its robust design and operation under parameter uncertainty is described. The pilot-scale run yielded a virus recovery of 86% and DNA and HCP clearances of 90% and 89%, respectively, without any fine-tuning of the operating parameters. This performance compares very favorably against that of single-column batch chromatography for the same volume of size-exclusion resin. To improve the robustness of the 2CSMB-SEC process, a general procedure for its design under parameter uncertainty is proposed under the framework of rigorous optimization theory. The optimal values of the operating variables are chosen only among candidates that are robust feasible, that is, that remain feasible for all possible variations of the uncertain parameters within their uncertainty intervals. In practice the nominal design problem is replaced by a worst-case design problem. The procedure was successfully employed to find the optimal robust values of the step durations when both the interparticle porosity and column volume are subject to two types of uncertainties. In one case the parameters are uncorrelated, uniformly distributed random variables, whereas in the other case they are normally distributed random variables. Moreover, in the second case the 2CSMB-SEC operating conditions were optimized to yield a given probability of success of satisfying both product quality specifications, say 95%, so that on average at least 95 out of 100 attempts to pack the two columns yield purified batches in which the product is within the specifications—this is achieved while maximizing the feed throughput

Purification of a hepatitis C vaccine candidate: Comparison between multi- column chromatographic processes operated in positive and negative mode

Given the increasing efficiencies in bioreaction and growing interest on complex biopharmaceutical products such as virus-like particles (VLPs), downstream processing (DSP) is becoming ever more relevant. Therefore, the biopharmaceutical industry is looking for alternative downstream strategies capable of improving purification yields whilst improving product quality and lowering costs. One of most promising improvements to DSP is to replace single-column batch operation by continuous, or semi-continuous, multi-column chromatography. We report on the development and comparison of two types of multi-column chromatographic systems aimed at the purification of Hepatitis C VLPs, produced using insect cell-based expression with recombinant baculovirus. The first process described herein is based on direct product capture using an anion exchange chromatographic media and subsequent elution with the modulation of ionic strength. By using a multi-column approach, one is able to overcome the limits of dynamic binding capacity characteristic of single-column batch processes, thus increasing the media capacity utilization. The second process reported is based on negative chromatographic purification. In this approach elution conditions are such that impurities should adsorb on the chromatographic media whereas the product of interest flows through the column. Both process approaches are subjected to a temporal arrangement of operations steps suchlike column equilibration, product application, production and regeneration. Volumetric productivity thus depends not only on the optimal scheduling of the referred steps, but also upon factors such as media capacity for the product and related impurities, operational flow-rates, and mechanical limitations of the systems used. The proposed analysis compares volumetric productivity, resin capacity utilization, equipment footprint and skid complexity for both purification strategies. Also we will demonstrate that the optimal design is a balance between the manufacturing scale, complexity and imposed product quality requirements

Estudo da variabilidade hospitalar das taxas de complicações de cuidados de saúde na população diabética no internamento

RESUMO - Contexto: O estudo das complicações de cuidados de saúde é cada vez mais relevante na investigação em saúde pública, na medida em que aumentam a morbilidade e a mortalidade e são responsáveis por um aumento substancial de custos diretos e indiretos na sociedade. Os objetivos definidos para este trabalho consistiram em estudar a variabilidade inter-hospitalar das taxas de complicações de cuidados durante o internamento na população diabética e identificar os seus fatores associados. Metodologia: Trata-se de um estudo transversal com 7347 doentes com diabetes mellitus tipo 2 (DMT2), como diagnóstico principal, com recurso à base de dados dos resumos de alta para os episódios de internamento no período de 01 janeiro a 31 de dezembro de 2015. Foram utilizados dois sistemas de classificação de doentes: os Grupos de Diagnóstico Homogéneo e o Disease Stanging. Na primeira abordagem usou-se a análise estatística descritiva onde se calculou a distribuição e as taxas de complicações de cuidados e o coeficiente de variação. Na segunda abordagem usou-se a regressão logística com recurso ao modelo de ajustamento pelo risco: as complicações de cuidados constituíram a variável dependente; o sexo, a idade, a gravidade da doença principal, as comorbilidades e respetivos níveis de gravidade, o número de dias de internamento, o hospital e o tipo de procedimento, as variáveis independentes. Resultados: As COC Outras infeções foram as mais prevalentes, com uma taxa de 31%, seguidas das COC Cardiorrespiratórias com 18,2% e das COC Urinárias que apresentaram uma taxa de 16,2%, havendo uma variação inter-hospitalar destas taxas. Os fatores associados como o sexo feminino (OR=2,248), a gravidade (3) da comorbilidade doença cerebrovascular (NEU04) (OR=1,737), a gravidade (1) da comorbilidade neoplasia maligna do cólon e reto (GIS27) (OR=2,417), bem como dez hospitais tiveram influência sobre a ocorrência de COC Urinárias. Os fatores como o sexo feminino (OR=1,223), as gravidades (1), (2) e (3) da comorbilidade pneumonia bacteriana (RES15), a gravidade (1) da comorbilidade rino, adeno e infeções do vírus Corona (RES24) (OR=58,566), treze hospitais e a gravidade (3) da doença principal (OR=2,008) tiveram influência sobre a ocorrência de COC Outras infeções. Constatou-se, ainda, que os fatores como as gravidades (1), (2) e (3) das comorbilidades RES15, doença arterial coronária sem revascularização (CVS11) e outras alterações do sistema respiratório (RES83), a gravidade (3) das comorbilidades insuficiência renal (GUS08), hipertensão essencial (CVS13) e doença cardíaca congestiva (CVS09) e as gravidades (2) e (3) da comorbilidade obesidade (NUT02) tiveram influência sobre a ocorrência de COC Cardiorrespiratórias. Conclusão: Os resultados indicam que as taxas COC Urinárias e COC Cardiorrespiratórias estão dentro dos valores apresentados por outros estudos recentes em países desenvolvidos, enquanto a taxa de COC Outras infeções é mais elevada em relação às taxas apresentadas por outros países industrializados. As taxas de COC não aumentam proporcionalmente com a dimensão dos hospitais, pelo que os hospitais de idêntica produção apresentam valores de taxas de COC distintas. Os diabéticos de gravidade (1) apresentam valores de taxa mais elevados do que os diabéticos de gravidade (2). As taxas de COC são mais elevadas no tipo de tratamento médico do que no cirúrgico. Os hospitais são fatores associados para as COC Urinárias e COC Outras infeções, mas não para as COC Cardiorrespiratórias. Estes resultados podem ser justificados por práticas clínicas distintas, pela adoção de protocolos de cuidados pouco seguros, entre outros. Os nossos resultados salientam a importância de implementar uma vigilância dirigida e medidas de reforço de prevenção e controlo das COC através da elaboração de modelos de segurança mais efetivos.ABSTRACT - Context: The study of complications care is more and more relevant in the investigation in the public health because increase the morbidity and mortality and could be responsible for a significant increase of direct and indirect costs in the society. Our aim was to evaluate the variability of the complications care rates during hospitalization in patients with diabetes mellitus and identify the influence of the intrinsic and extrinsic risk factors in these complications. Methodology: Were conducted a cross-sectional stud on 7347 patients with type 2 diabetes mellitus as the principal diagnostic with hospital discharge in 2015. The information of the database of the summaries of medical discharge was used, as well as the classification system of the patients: The Groups of Homogeneous Diagnostic and the Disease Staging. The first approach used the descriptive statistics analysis to calculate the distribution of complication care rates and the coefficient variation to determinate the inter-hospital variation. The second approach used the logistic regression using the risk-adjustment model: the COC, individually, was the dependent variable; sex, age, the severity of the principal disease, the severity of the comorbidities, number of hospitalization days, hospital and type of procedure, were independent variables. Results: The rate of COC Infections, other were the most prevalent with 31%, followed by COC Cardiorespiratory with 18.2% and COC Urinary which presented a rate of 16.2%, with a significant inter hospital variation. The risk factors, such as female gender (OR=2.248), the stage (3) of comorbidity Cerebrovascular disease (NEU04) (OR=1.737) and the stage (1) of comorbidity Malignant neoplasm of colon and rectum (GIS27) (OR=2.417), as well as ten hospitals had influence over the occurrence of COC Urinary. Factors such as female gender (OR=1.223), the stages (1), (2) and (3) of comorbidities Bacterial pneumonia (RES15), the stage (1) of comorbidity Rhino, adeno and infections of the Corona virus (RES24) (OR=58.566), thirteen hospitals and the stage (3) of the principal disease (OR=2.008) influenced the occurrence of COC Infection, other. It was also found that factors such as stages (1), (2) and (3) of comorbidities RES15, Coronary artery disease without revascularization (CVS11) and Other alterations of the breathing system (RES83), the stage (3) of comorbidities Renal insufficiency (GUS08) and Essential hypertension (CVS13) and Congestive heart failure (CVS09) and stages (2) and (3) of comorbidity Obesity (NUT02) influenced the occurrence of COC Cardiopulmonary. Conclusion: The results indicate that the COC Urinary and COC Cardiopulmonary rates are within the values presented by other recent studies in developed countries. The COC Infections, other rate is higher than rates presented by other industrialized countries. Hospitals with the same production show different values of complication care rates. The complication care rates do not increase proportionally with the hospital production. Diabetics of stage (1) had higher rate values than diabetics of stage (2). The COC rates are higher in the type of medical treatment than in the surgical one. The severity of illness is an independent risk factor of COC Urinary. The hospitals are an independent risk factor for the COC Cardiopulmonary, but not for COC Urinary and COC Infections, other. These results can be justified by different clinical practices, the adoption of unsafe care protocols, and among others. These findings lend support to the importance of implementing a targeted surveillance and development effective models of patient safe in COC

Multi-column chromatographic purification of influenza virus-like particles

Seasonal Influenza occurs all over the world and causes approximately 500 million cases of infection and up to 500 thousand deaths annually. The most effective way to prevent the disease is throughout vaccination. However, the constant antigenic drift on the influenza virus implies an annually vaccine update with high inherent costs. A new generation of virus-like particles (VLPs) vaccines, that have the ability to stimulate the production of broad antibody response to different Influenza strains, is a promising approach to solve this problem. VLPs have become a promising solution for influenza pandemics as well. The high attractiveness of VLPs has led to an increasing interest in the development of VLP purification processes as it often accounts for the major production costs. Therefore, it is mandatory to improve downstream processing (DSP) trains, not only to increase the efficiency of the existing processes but also to develop new unit operations capable of coping with the stringent regulatory requirements. One of the most promising improvements to DSP is to replace single-column batch operation by continuous, or semi-continuous, multi-column chromatography. The process herein described is based on the optimal scheduling of the operations steps characteristic of a single-column bind and elute operation such as equilibration, product application, production and regeneration applied to a train of two columns. In fact, a simple serial connection of two columns, during the product application step, directing the effluent of the first column in the train to an adjacent one, allows the capture of the mass transfer zone. This setup modification not only avoids product loss but also greatly increases the capacity utilization of the chromatographic media by achieving column saturation. We report on the development of a multi-column chromatographic process aimed at the purification of influenza VLPs, produced using insect cell-based expression with recombinant baculovirus. The inherent potential to improve process efficiency and economics, providing a powerful and flexible alternative to conventional batch chromatography will be demonstrated highlighting the impact of factors such as manufacturing scale, the complexity of the experimental setup and imposed product quality requirement

Inpatient Mortality in People With Type 2 Diabetes: A Cross-sectional Study

Objective: This study aimed to estimate inpatient mortality rate for diabetes and identify its associated factors. Materials and methods: This is a cross-sectional study. The population was comprised between January 1 and December 31, 2019 in 32 public hospitals in Portugal, using summary hospital discharge data. We used both the Disease-Related Diagnosis Groups and the Disease Staging. Patients were grouped into survivors and non-survivors, and inpatient mortality was compared using competing event regression. Results: A total of 7980 patients were admitted with type 2 diabetes mellitus, there were 747 (10.3%) non-survivors. The advanced age (OR = 1.772; 95% CI 1.625–1.932), the stage (3) severity of type 2 diabetes mellitus (OR = 4.301; 95% CI 2.564–7.215), comorbid lung, bronchial or mediastinal malignant neoplasm (OR = 5.118; 95% CI 2.222–11.788), comorbid bacterial pneumonia (OR = 3.214; 95% CI 2.539–4.070), other respiratory system disorders (OR = 2.187; 95% CI 1.645–2.909), comorbid rhino-, adeno- and coronavirus infections (OR = 1.680; 95% CI 1.135–2.488) were determinants for inpatient mortality. Conclusions: Elderly patients with diabetes with micro- and macrovascular complications of the disease, who have bacterial pneumonia and who enter the emergency department are those who have a lower survival rate

Advancing downstream purification of cell and gene therapy medicinal products

The advent of advanced therapies in the biopharmaceutical industry has moved the spotlight into complex products such as viral vectors or stem cells, holding great promise in a myriad of clinical targets. Currently, the challenge for a widespread application of these new biopharmaceuticals is the development of cost-effective bioprocesses while maintaining product\u27s bioactivity and quality attributes. This presentation will focus on the latest advancements on downstream purification of cell and gene therapy medicinal products, supported by process innovation and the flexibility of old, but robust technologies such as tangential flow filtration (TFF) and chromatography. Improvement of purification yields of virus based biopharmaceuticals can be achieved through the rational development of alternative strategies, combining different modes of operation, such as flow through purification or multi-column chromatography, together with the recent developments of chromatographic media and fundamental understanding of the adsorption phenomena as reported for the case of gene therapy medicinal products. Critical quality attributes of cell based medicinal products cannot be compromised by the processing route chosen. The use of the already established TFF technology has the potential to improve the purity of cell based products, with the evaluation of critical process parameters of cell concentration and washing being of paramount importance. The purity of such products can also be incremented with the use of negative mode expanded bed adsorption chromatography with a new multimodal prototype matrix based on core–shell bead technology as demonstrated for the case of human mesenchymal stem cells. In summary, the advancement of the purification of complex biopharmaceutical entities, such as the ones here reported, can be described as an incremental process, but still with space for inn

Towards an integrated continuous manufacturing process of adeno- associated virus (AAVs)

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Bioprocess engineering of insect cells for accelerating vaccines development

The majority of novel protein-based biologics require animal cell technology for discovery and production. The costs and complexity associated to these production platforms are commonly high making mandatory further improvements in product yields. Technological breakthroughs and/or new producer hosts allied to classical systems/molecular biology tools and engineering methodologies are therefore necessary for accelerating biologics manufacturing process, including vaccines. Two case studies will be presented where bioprocess engineering assisted/accelerated vaccines development. In the first case study, the aim was to generate an Influenza VLP in which the surface antigens are presented in their native conformation as membrane-bound proteins and are comprised of several hemagglutinin (HA) variants specifically designed to target B cells capable of mounting broad neutralising antibodies. The platform herein adopted for production of enveloped Influenza VLP enclosed certain drawbacks, namely (1) instability of baculovirus vectors encompassing multiple HA genes, and (2) low expression levels and recovery yields. To tackle these issues, a set of bioprocess engineering schemes were designed and subsequently implemented, which included (1) combination of stable and baculovirus-mediated expression of HA in insect High Five cells for production of difficult to express multi-HA VLPs (pentavalent VLP of H3 subtype), (2) DoE for identifying best infection strategy and evolutionary engineering of insect cells phenotype, and (3) development of a scalable, “universal” and “All-Filtration” purification platform of Influenza VLPs. In the second case study, the aim was to develop a fast and flexible insect cell platform for production of enveloped VLPs pseudo-typed with membrane proteins of interest. Stable insect cell lines have been successfully generated using site-specific gene integration based on flipase-mediated cassette exchange (FMCE) technology. Influenza M1 and HIV Gag proteins were evaluated as scaffolds, and proof-of-concept demonstrated using two membrane proteins, the Influenza HA protein (e.g. for vaccines) and the human beta-2 adrenergic receptor (e.g. for drug screening or antibody discovery). Bioprocess engineering schemes have been designed (adaptive laboratory evolution to hypothermic culture conditions and supplementation with productivity enhancers), allowing to improve Gag-VLP production in the developed stable insect cells. Overall, the insect cell platforms and bioprocess engineering strategies herein assembled have the potential to assist/accelerate vaccines development. Acknowledgments: This work was supported by European Commission (Project EDUFLUVAC, Grant nr. 602640) and by Portuguese “Fundação para a Ciência e a Tecnologia” through the following programs: FCT Investigator Starting Grant (IF/01704/2014), Exploratory Research and Development Project EXPL/BBBBIO/1541/2013, and PhD fellowships SFRH/BD/86744/2012 and SFRH/BD/90564/201

Continuous purification of hepatitis C virus-like particles by multi-column chromatography

Novel biopharmaceutical products, such as virus-like particles (VLPs) and viral vectors, constitute a challenging task for downstream processing (DSP). Recoveries achieved to reach required purities are significantly inferior compared to more common antibody and other recombinant processes. Therefore, the biopharmaceutical industry is looking for alternative downstream strategies capable of improving purification yields and decreasing cost while maintaining product quality. One of many possible improvements to DSP is to replace single-column batch operation by continuous, or semi-continuous, multi-column chromatography. A single-column batch chromatographic operation used for capture is limited by the dynamic binding capacity (DBC) of the target product. For high-value products, chromatographic columns are normally loaded to less than 1% of DBC underutilizing the resin\u27s capacity. Increasing capacity utilization leads to significant resin cost savings, particularly relevant in the case of capture with expensive affinity materials. Multi-column processes have been shown to improve process efficiency and economics, providing a powerful and flexible alternative to conventional batch chromatography. In fact, a simple serial connection of two chromatographic columns, where the effluent of the first column of the train is directed to the inlet of the second column, allows that in a loading step the breakthrough of the first column is captured on the second bed, thus avoiding product loss. After saturation, the first column can be subjected to the normal processing steps of a batch chromatographic operation while loading is resumed in the adjacent bed. Moreover, this simple setup modification allows not only to extend the utilization of the resin’s capacity, overcoming the aforementioned issues, but also to benefit from the counter-current flow between the mobile and the stationary phases, which optimizes the driving force for mass transfer throughout the overall trajectory of the two phases. We report the development of a continuous chromatographic process for the purification of Hepatitis C VLPs (HCV-VLPs), produced using insect cell-based expression with recombinant baculovirus. A library of novel anion exchange resins with different ligand densities was evaluated for improved binding and release of the target product and impurity clearance in batch operation. A model-based approach for a smooth transition from a single-column batch process to a continuous multi-column operation is demonstrated and the scheduling of periodic events of the process cycle is analyzed. The contribution of column overloading, counter current operation and faster flow rates to recovery improvements compared to batch is discussed. Ultimately, both purification strategies, batch and continuous, are compared not only in terms of volumetric productivity, resin capacity utilization and footprint reduction, but also to indicate whether actual performance can be improved by continuous operation