Development of an innovative real-time assay for antimalarial sensitivity testing

Tese de doutoramento, Ciências Biomédicas (Microbiologia e Parasitologia), Universidade de Lisboa, Faculdade de Medicina, 2017Antimalarial drug resistance has always been an obstacle in the fight against malaria. Malaria parasites have developed resistance to most available antimalarial drugs and, more recently resistance to artemisinins, the first-line treatment for malaria, is emerging and spreading in Southeast Asia. Artemisinin resistance is characterized by delayed parasite clearance times observed in malaria patients. For now this resistance is considered to be partial by the WHO and artemisinin combination therapies remain as the mainstay of antimalarial treatment. In vitro assays are of paramount importance to detect and monitor drug resistance. Several in vitro drug assays exist, however their inherent characteristics, such as the use of radioactive or expensive reagents and their long turn-around times limit their application. The project developed in the context of this thesis aimed to develop a novel drug assay for Plasmodium spp. that would overcome some of the limitations of currently available drug assays. The underlying idea was to use hemozoin, a crystal produced by malaria parasites, to measure their own growth or maturation which would allow to detect drug effects. The hemozoin content increases as parasites mature inside the erythrocytes. Thus, hemozoin constitutes an optimal parasite maturation biomarker. Moreover, it is a birefringent crystal, and as such it is able to depolarize light. The resulting light depolarization can be easily detected by optical methods such as flow cytometry. It was previously shown by our laboratory that in a rodent model of malaria depolarization caused by hemozoin inside infected erythrocytes could be detected using a simple flow cytometric set-up. Moreover, the inhibitory effect of commonly used antimalarial drugs could also be determined after only 6-8 hours of incubation. The main objectives of the work developed during this thesis was to further develop the flow cytometric detection of hemozoin assay using P. falciparum in vitro cultures and to assess its performance ex vivo, in the field, using samples from malaria patients. A benchtop flow cytometer (Cyflow SL) was modified to allow the detection of light depolarization and it was used for all studies. Other commercially available cytometers (MoFlo, Accuri C6, Attune) were also easily adapted to detect light depolarization, showing that the measurement of this additional parameter can be accomplished in different instruments. In vitro cultures of P. falciparum were established and allowed to further investigate the potential of this novel method. Ring-stage synchronized cultures of P. falciparum were incubated with several antimalarial drugs (chloroquine, mefloquine, quinine, artemisinin, artesunate and pyrimethamine). Analysis of depolarizing events, corresponding to parasitized erythrocytes containing hemozoin, allowed the detection of parasite maturation. Furthermore, chloroquine resistance and the inhibitory effect of all antimalarial drugs tested, except for pyrimethamine, could be determined as early as 18 - 24 hours of incubation. The 50% inhibitory concentrations (IC50) obtained at 24 hours of incubation were comparable to previously reported values. However, these values were most of the times higher than the ones obtained with the already validated HRP2 ELISA assay. Indeed, IC50 values may differ considerably between assays. Different assays measure different parameters to assess parasite growth, at different time-points. Moreover, variations in parasite density and hematocrit as well as the stage-dependent action of antimalarial drugs, may influence these values. Altogether, explaining the differences in IC50 values that are commonly observed. The performance of the hemozoin detection assay in the field was assessed during a 6- month trial performed in Gabon, a malaria endemic country. The trial was conducted in the Centre de Recherches Médicales de Lambaréné – Albert Schweitzer Hospital. On site, an existing flow cytometer (Cyflow SL) was modified to detect light depolarization caused by hemozoin. A total of 46 samples from malaria patients were analyzed during this study. Blood samples were incubated with increasing concentrations of chloroquine, artesunate and artemisinin. The percentage of depolarizing cells was used as maturation indicator and measured at 24, 48 and 72 hours of incubation to determine parasite growth and drug effects. Analysis of ex vivo cultures of parasites obtained from blood samples of malaria patients showed four different growth profiles. The flow cytometric detection of hemozoin allowed to detect drug effects in 39/46 (85%) of samples. In 25 samples drug effects were measurable at 24 hours. In the remaining 14 samples parasite maturation was delayed, and thus drug effects were only detected at 48 hours of incubation. Obtained IC50 values showed that chloroquine-resistant parasites were still common and present in Lambaréné, Gabon but they were fully sensitive to artesunate and artemisinin. Finally, the usefulness of the hemozoin detection assay in the investigation of artemisinin resistance in vitro was also assessed. Artemisinin-resistant (MRA-1240) and sensitive (MRA-1239, 3D7) strains were cultured in vitro. Parasite maturation was determined based on the flow cytometric detection of hemozoin-containing cells. Two different drug assays were performed: 1) standard drug assay: where ring-stage parasites were continuously incubated with increasing concentrations of dihydroartemisinin (DHA) for 48 hours; and 2) pulse assay: where tightly ring-stage synchronized parasites were incubated with a single high-dose (700 nM) of DHA for 6 hours. Results showed that at 24 hours of incubation artemisinin-resistant parasites had increased IC50 values, in comparison to the artemisinin-sensitive strains (15 nM and 8 nM, respectively). Moreover, when parasites were exposed to a high-dose of DHA for 6 hours, increased survival rates associated with artemisinin resistance could be detected after only 30 hours of incubation. Interestingly, it was also observed that artemisinin-resistant parasites do not seem to enter dormancy, as it has been previously suggested by others. Microscopic assessment performed after 72 hours of incubation showed that parasites that survived to a 6-hour exposure to DHA were very close in terms of parasite development to the ones found in the drug free control. Further investigation using more artemisininresistant strains is required to determine whether increased IC50 values correlate with the delayed parasite clearance times observed in the patients; and if the underlying mechanisms of artemisinin resistance is or not related to dormancy. Overall, the work presented in this thesis shows that hemozoin detection by flow cytometry is an alternative, reagent-free and rapid drug assay that overcomes some of the limitations of currently available drug assays for P. falciparum. Moreover, it may also be a useful tool in the study of artemisinin resistance both in culture-adapted strains and, possibly in strains obtained directly from patients. Importantly, this work paves the way for the development and investigation of better tools to assess drug effects and monitor drug resistance in Plasmodium spp. Several novel hemozoin detection platforms are available or under development and should definitely be further explored for their potential to be used as antimalarial drug assays. Furthermore, combination of hemozoin detection with the measurement of other parameters, such as DNA and RNA content and parasite viability may even provide additional important information to reliably determine the developmental stage and metabolic status of parasites and, consequently, detect drug effects. Hopefully, this would lead to the development of an optimal antimalarial drug assay that could play an important role in the fight against malaria

Malarial pigment (hemozoin): flow cytometric detection of hemozoin in infected erythrocytes to monitor drug-effects

Tese de mestrado. Biologia (Microbiologia Aplicada). Universidade de Lisboa, Faculdade de Ciências, 2010A malária é uma doença que tem sido uma preocupação constante para a humanidade ao longo de séculos, continuando a ser responsável por cerca de 1 milhão de mortes por ano.84 O impacto da malária é elevado estende-se muito além das taxas de mortalidade e morbilidade associadas.20 A malária em humanos é causada por cinco espécies de parasitas: Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae e Plasmodium knowlesi. O parasita Plasmodium falciparum é responsável pela maioria dos casos em África e dos casos de malária severa. Estas cinco espécies diferem na distribuição geográfica, características microscópicas, características clínicas e no potencial para desenvolver resistência aos anti-maláricos. O ciclo de vida do parasita é complexo e envolve um vector e um hospedeiro vertebrado. Durante o ciclo de vida, no hospedeiro, existem duas fases principais: fase hepática e a fase sanguínea. Durante a fase sanguínea o parasita invade eritrócitos, desenvolve-se e multiplica-se no seu interior. Esta fase é a responsável pela sintomatologia associada à infecção por malária. Durante o desenvolvimento do no interior dos eritrócitos o parasita degrada a hemoglobina para obter aminoácidos e para regular a pressão ósmotica.28 Como consequência desta degradação é produzido heme livre. Este, tal como acontece em organismos superiores, é tóxico para o parasita.78 Para ultrapassar este obstáculo o parasita desintoxica o heme livre transformando-o em hemozoína (Hz), também denominado por pigmento malárico.39 O conteúdo de Hz aumenta à medida que o parasita matura constituindo, assim, um óptimo indicador de crescimento.7 Vários anti-maláricos interferem com a produção de Hz. Fármacos que contêm quinolina, como a cloroquina, quinino, mefloquina, amodiaquina e halonfantrina têm como principal mecanismo de acção a inibição da produção de Hz, levando à morte do parasita, como uma consequência da acumulação de heme livre tóxico.28,69,78 Outro grupo de fármacos que também pode estar envolvido na inibição da produção de Hz é eventualmente a artemisina e os seus derivados.55 Apesar de existirem algumas provas que apontam para o envolvimento da Hz, o mecanismo de acção deste fármacos é um tema controverso, e ainda não está completamente esclarecido. Nas últimas décadas tem-se vindo a observar um aumento da resistência a determinados antimaláricos. A resistência aos anti-maláricos é um grave problema de saúde publica, uma vez que está associado à propagação da malária a novas áreas e ao reaparecimento em regiões onde fora erradicada.6 Perante este cenário torna-se imprescindível ter conhecimento das susceptibilidades do parasita aos diferentes anti-maláricos. Actualmente existem diversos testes de sensibilidade. Basicamente, podemos agrupá-los em: ensaios in vivo e ensaios in vitro. Estes testes são úteis para definir guidelines para politicas de saúde pública, são ferramentas essenciais para o desenvolvimento de novos fármacos e para a epidemiologia da resistência a anti-maláricos. Por outro lado possuem diversas limitações, como o longo turn-around time (30 h - 96 h), o uso de equipamento sofisticado e de reagentes dispendiosos e difíceis de adquirir. Estas limitações contribuem para o desenvolvimento de novos métodos capazes de as ultrapassar. Este projecto tem como principal objectivo o desenvolvimento de um teste de sensibilidade com base na detecção de Hz por citometria de fluxo. Devido às propriedades físicas da Hz (birrefringente), esta pode ser detectada por métodos ópticos, como por citometria de fluxo. Recorrendo a um citómetro modificado foi possível detectar eritrócitos infectados (Ei) contendo diferentes quantidades de hemozoína. Amostras de sangue, colhidas de ratinhos infectados com Plasmodium berghei, foram incubadas (in vitro) com vários anti-maláricos (cloroquina, quinino, mefloquina, artemisinina e pirimetamina), durante 24 horas de incubação. Analisando a percentagem de Ei contendo uma elevada quantidade de Hz, denominadas “eritrócitos infectados que depolarizam muito” (Ei-dm), foi possível detectar o efeito inibitório dos fármacos. A avaliação da percentagem de Ei-dm, possibilitou a detecção de um aumento da quantidade de Hz no interior dos Ei, consequência da maturação do parasita. Contrariamente, nas amostras tratadas com cloroquina, quinino, mefloquina e artemisinina não foi detectado nenhum aumento, indicando que os fármacos inibiram o crescimento do parasita e, consequentemente, não foi produzida mais Hz. Curiosamente nas amostras tratadas com cloroquina detectou-se uma diminuição acentuada na percentagem de Ei-dm nas primeiras horas de incubação. Este resultado inesperado revelou-se ser consequência da agregação dos cristais de Hz. Este fenómeno de agregação, denominado “clumping”, foi descrito anteriormente, onde na presença de cloroquina os cristais de Hz formam um agregado.76 A detecção de Ei-dm resulta da avaliação da depolarização da luz que é dispersada lateralmente (depolarized side scattered light: depolarized-SSC). Este parâmetro, SSC, é um indicador da granulosidade celular.64 Deste modo, é muito provável que o depolarized-SSC possa também ser influenciado pela granulosidade celular. Neste caso, se um Ei possuir cristais de Hz dispersos e outro tiver um agregado de Hz, o sinal de depolarized-SSC será menor no Ei com um agregado de Hz no seu interior, fazendo com que estes Ei deixem de pertencer à população de Ei-dm. Justificando-se, assim, a diminuição inicial na percentagem de Ei-dm nas amostras tratadas com cloroquina. Relativamente à pirimetamina nenhum efeito foi detectado. Este resultado não foi de todo inesperado, uma vez que a pirimetamina actua numa fase tardia da maturação do parasita (no esquizonte).50 Deste modo, o efeito da pirimetamina é apenas detectado após um ciclo de replicação, ou seja, nas gerações seguintes de parasitas. Devido ao modelo experimental usado (murganhos infectados com Plasmodium berghei), a cultura de Ei testada apresenta apenas uma geração de parasitas. Isto porque P. berghei in vitro não é capaz de romper os eritrócitos e, consequentemente, não re infecta novos eritrócitos.32 A artemisina é um fármaco cujo mecanismo de acção permanece desconhecido. Possíveis mecanismos como o envolvimento na produção de hemozoína55 e a alteração das membranas do parasita26, têm vindo a ser sugeridos nos últimos anos. Os resultados obtidos demonstram que na presença de concentrações mais elevadas de artemisina (129 nM e 259 nM) há um decréscimo gradual da percentagem de Ei-dm, a partir das 2 horas de incubação. Este resultado pode sugerir que tal como acontece com a cloroquina, mas não tão rapidamente, o pigmento pode começar a agregar-se, ou então, que o fármaco pode provocar a lise dos Ei. Nenhuma destas hipóteses pôde ser confirmada porque durante esta experiência não foram adquiridas contagens absolutas da concentração de eritrócitos presentes nas amostras. A utilização de estirpes de parasitas que expressam GFP (green fluorescent protein) para o screening de anti-maláricos e determinação da do parasita a diferentes compostos sensibilidade foi descrita tanto para P. falciparum como para P. berghei. 60,61 Na maioria das experiências realizadas ao longo deste projecto foram usadas estirpes de parasita que expressam GFP para que fosse possível comparar os resultados obtidos através destas duas abordagens (detecção de parasitas que expressam GFP ou detecção de Ei-dm). O efeito do fármaco foi detectado com uma maior antecedência e evidência através da análise da percentagem de Ei-dm. Concluindo, a detecção por citometria de fluxo de Ei contendo diferentes quantidades de Hz permitiu a avaliação do efeito inibitório de todos os anti-maláricos testados, à excepção da pirimetamina, em apenas 4 horas de incubação. Apesar dos resultados do projecto demonstrarem o potencial deste ensaio é preciso ter em conta que o objectivo principal é desenvolver um teste de sensibilidade para Plasmodium spp. responsáveis pela malária em humanos e, para isso, uma cultura contínua de P. falciparum está a ser estabelecida. No futuro será também importante testar estirpes de P. falciparum resistentes de modo a optimizar o ensaio. Posteriormente, será também de interesse a recolha e análise de amostras de sangue de doentes com malária num país onde esta doença é endémica.Malaria remains the most important parasitic disease. The emergence and spread of drug resistant Plasmodium falciparum is a subject of great concern and requires monitoring of the parasite susceptibility. Currently there are several sensitivity assays for Plasmodium falciparum but they are associated with important limitations, such as: long turn-around times (30 h - 96 h), use of expensive equipment and sophisticated or labour-intensive methodologies. Malaria parasites produce hemozoin (Hz), in order to detoxify heme after hemoglobin degradation. Hz content increases during parasite maturation, thus constituting an optimal growth indicator. Due to its physical property (birefringence) it can be detected by optical methods, like flow cytometry. Using a purpose built flow cytometer, it was possible to detect infected red blood cells (iRBCs) containing different amounts of Hz. The principal goal of this project is to develop a rapid and reliable sensitivity assay. This kind of assay will surely be a great tool for the assessment of parasite susceptibility and for the high throughput screening of new compounds. Blood from Plasmodium berghei infected mice was incubated (in vitro) with several antimalarial drugs (chloroquine, quinine, mefloquine, artemisinin and pyrimethamine), over 24 hours. Analysing the percentage of iRBCs containing high amounts of Hz, denominated as highly depolarizing RBCs (hdRBCs), it was possible to detect the inhibitory effect of several drugs. In the untreated samples the amount of Hz within iRBCs increased, as assessed by the percentage of hdRBCs, due to parasite maturation. Contrary to this, no increase was detected in samples treated with chloroquine, quinine, mefloquine and artemisinin. Pyrimethamine effect could not be detected. The inhibitory effect of all antimalarial drugs tested, excluding pyrimethamine, could be detected already after 4 hours of incubation. These preliminary results suggest that this method could be developed into a rapid, objective and automatable sensitivity test, without the need for reagents

Invasive hornets on the road: motorway-driven dispersal must be considered in management plans of Vespa velutina

Understanding the mechanisms that potentiate the dispersion of an invasive species is essential to anticipate its arrival into new regions and to develop adequate management actions to minimize damage to biodiversity and society. One of the most successful invaders in Europe, the yellow-legged hornet (Vespa velutina), is dispersing through self-diffusion and jump dispersal. Using information on species occurrence in Portugal from 2013 to 2018, this study aimed to understand the range expansion trajectory of V. velutina and to identify the role of climate, landscape and anthropogenic variables on the two mechanisms of spread. We found that in Portugal the invasion is proceeding faster southwards (45 km/year) along the Atlantic coast than eastwards (20 km/ year) where the climatic suitability gradient is more compressed, with jump dispersal playing an important role in this difference and in the acceleration of the invasion process. Dispersal by diffusion was best explained by the annual range of temperature and precipitation of the wettest month, with distance to shrub land also having an important role. Additionally, jump dispersal appeared to be facilitated by motorways, hinting at the role of human-mediated dispersal. Indeed, the number of nests that resulted from this dispersive mechanism were significantly closer to motorways than expected by chance. To prevent the dispersal of V. velutina into Mediterranean regions, and in addition to a special attention to the advancing front, early monitoring programs should also target a buffer zone on both sides of motorways, and at freight shipping hubs.info:eu-repo/semantics/publishedVersio

EARLY ACCESS TO THE INTEGRATED EMERGENCY MEDICAL SYSTEM: A STUDY WITH CHILDREN 6-12 YEARS OLD

Introduction: One of the most incriminating in human life is the occurrence of an unexpected cardiac arrest. Despite advances in the field of cardiopulmonary resuscitation, the risk of death remains between 50-80% (Buist et al., 2002). The increased survival rate of a patient in cardiac arrest depends on the timing of the start, the local maneuvers Basic Life Support, until the arrival of rescue more differentiated. Early access to the Integrated Emergency Medical System (IEMS) is therefore a decisive stage. Methods: This study sought to understand the ability of children 6-10 years old: recognizing a person unresponsive to stimuli and unventilated; accurately identify their place of residence; indicate the national emergency number. The research was conducted in a group of schools in the district of Portalegre (Portugal), students in 9 classes from basic education, a total of 122 students. For data collection we designed a questionnaire, applied in between 11 and 15 June 2012. Results: The results point to an illiteracy on evaluative component in response to stimuli and ventilation presence. Most students reveal not able to distinguish whether it is the presence of a sleeping person or someone who does not ventilate. Students participating in the study, mostly, do not know their full address. The national emergency number is unknown by most students. Conclusions: These results show the urgent need to empower students ages these skills to the drive level of IEMS and early recognition of unconsciousness and lack of ventilation person. It is suggested, accordingly, that teachers are adequately prepared to train students in these skills, decisive to increase survival rates in the event of cardiac arrest

Recognition of the epidemiological significance of Neisseria meningitidis capsular serogroup W135 in the Rio de Janeiro region, Brazil

Neisseria meningitidis retains its ability to cause endemic and hiperendemic disease in human population living in any environment, as well as localized outbreaks and massive epidemics in civilians and military personnel. In Rio de Janeiro it has been reported in the 1990s as prolonged outbreak of serogroup B and at least one epidemic of serogroup C was well defined, both demanding quick action by the Public Health authorities. We report here the emergence of serogroup W135 meningococcal disease causing endemic and case cluster in Rio de Janeiro during the first years of this new century

Human activity recognition for an intelligent knee orthosis

Dissertação para obtenção do Grau de Mestre em Engenharia BiomédicaActivity recognition with body-worn sensors is a large and growing field of research. In this thesis we evaluate the possibility to recognize human activities based on data from biosignal sensors solely placed on or under an existing passive knee orthosis, which will produce the needed information to integrate sensors into the orthosis in the future. The development of active orthotic knee devices will allow population to ambulate in a more natural, efficient and less painful manner than they might with a traditional orthosis. Thus, the term ’active orthosis’ refers to a device intended to increase the ambulatory ability of a person suffering from a knee pathology by applying forces to correct the position only when necessary and thereby make usable over longer periods of time. The contribution of this work is the evaluation of the ability to recognize activities with these restrictions on sensor placement as well as providing a proof-of-concept for the development of an activity recognition system for an intelligent orthosis. We use accelerometers and a goniometer placed on the orthosis and Electromyography (EMG) sensors placed on the skin under the orthosis to measure motion and muscle activity respectively. We segment signals in motion primitives semi-automatically and apply Hidden-Markov-Models (HMM) to classify the isolated motion primitives. We discriminate between seven activities like for example walking stairs up and ascend a hill. In a user study with six participants, we evaluate the systems performance for each of the different biosignal modalities alone as well as the combinations of them. For the best performing combination, we reach an average person-dependent accuracy of 98% and a person-independent accuracy of 79%

Psychometric Properties of the Brazilian Version of GOHAI among Community-Dwelling Elderly People

publishedVersio

Canine hidrotherapy

A hidroterapia é um recurso que pode ser utilizado como meio terapêutico nos animais. Este artigo de revisão bibliográfica tem como finalidade dar a conhecer as propriedades físicas da água, os seus benefícios e indicações no tratamento de cães. São traçadas algumas considerações sobre o plano de tratamento dos cães e expostas as várias modalidades existentes, tal como apresentados alguns dos exercícios terapêuticos que se podem realizar e equipamentos utilizados. Finalmente são abordadas as questões relacionadas com as precauções e contraindicações da hidroterapia, assim como os cuidados com o tratamento da água. Em conclusão, sugere-se a realização de mais investigação nesta área, em que veterinários e fisioterapeutas devem colaborar em conjunto, com vista a beneficiar o melhor amigo do Homem, o cão.ABSTRACT - Hydrotherapy is a feature that can be used for therapeutic purposes in animals. This literature review article aims to make known the physical properties of water, its benefits and indications for the treatment of dogs. This review makes considerations about the treatment plan of dogs and presents the various therapeutic exercises and equipment currently available. We discuss the issues, precautions and contraindications of hydrotherapy, as well as water treatment requirements. In conclusion, it is recommended to carry out more research in this area, where veterinarians and physiotherapy professionals should work together in order to benefit man’s best friend, the dog