Utilize este identificador para referenciar este registo: http://hdl.handle.net/10451/28478
Título: Development of an innovative real-time assay for antimalarial sensitivity testing
Autor: Rebelo, Maria Sousa, 1986-
Orientador: Hänscheid, Thomas, 1964-
Mota, Maria Manuel Dias da, 1971-
Palavras-chave: Teses de doutoramento - 2017
Resistência a medicamentos
Citometria de fluxo
Data de Defesa: 2017
Resumo: Antimalarial 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.
Descrição: Tese de doutoramento, Ciências Biomédicas (Microbiologia e Parasitologia), Universidade de Lisboa, Faculdade de Medicina, 2017
URI: http://hdl.handle.net/10451/28478
Designação: Doutoramento em Ciências Biomédicas
Aparece nas colecções:FM - Teses de Doutoramento

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