Evaluation of novel molecular markers for monitoring drug resistence in Plasmodium falciparum malaria

Tese de mestrado, Microbiologia Clínica, Faculdade de Medicina, Universidade de Lisboa, 2010The human malaria parasite Plasmodium falciparum is acquiring resistance to most drugs it has encountered, including the recently deployed Artemisinin Combination Therapy, on which much hope has been laid. Molecular markers for monitoring the evolution of resistance are, therefore, urgently required. Our group has recently made use of a rodent malaria model to identify a number of novel genetic markers of antimalarial drug resistance, namely a clathrin mu adaptor gene (pfcmu) involved in artemisinin resistance and an amino acid transporter gene (pfaat1) underlying chloroquine resistance. The main aim of this thesis was to characterize and evaluate the contribution of the above genes to drug resistance in natural parasite populations of P. falciparum isolates from three endemic areas: Rwanda, Democratic Republic of Sao Tomé & Principe (DRSTP) and Brazil. The global diversity of pfcmu and pfaat1 was determined, resulting in the identification of several polymorphisms. The pfaat1 gene appears to be highly conserved and no correlations were found between this gene and the in vitro resistance to 4-aminoquinolines. In contrast to pfaat1, the pfcmu gene was genetically diverse, with nine Single Nucleotide Polymorphisms and three different insertions identified across all isolates inspected. Samples could be grouped in to fourteen different pfcmu haplotypes, whose diversity was higher in both African sites than in Brazil (Hd = 0.964 ± 0.077, 0.750 ± 0.139 and 0.250 ± 0.180 in Rwanda, DRSTP and Brazil, respectively). Some of the identified polymorphisms showed geographical specificity. We found a significant association between a pfcmu G479A genotype in Rwanda samples and the in vitro sensitivity to dyhidroartemisinin (p = 0.0207). These constitute new findings to suggest that polymorphisms in pfcmu can be involved in P. falciparum defence mechanisms against artemisinin derivatives. Thus, further assessment of the gene in artemisinin responses is a top priority, in the context of effective surveillance of artemisinin resistance.A resistência do parasita Plasmodium falciparum aos fármacos é um dos principais obstáculos a uma contenção eficiente da malária. Os compostos utilizados para o combate a esta doença têm perdido sua eficácia ao longo dos anos, incluindo a artemisinina e os seus derivados, recentemente indicados como promissores no tratamento da malária. De facto, foram descritos recentemente os primeiros casos de resistência in vivo a estes compostos em parasitas de malária humana. Consequentemente, a identificação de marcadores moleculares de resistência a estes fármacos, previamente a um alastramento da resistência, apresenta-se como uma estratégia essencial. Recentemente, fazendo uso de um modelo de malária de roedores (Plasmodium chabaudi), o nosso grupo identificou um número de determinantes genéticos de resistência a diferentes antimaláricos, nomeadamente, o gene pfcmu, que codifica uma subunidade mu do complexo adaptador de clatrina e se relaciona com resistência aos derivados da artemisinina e o gene pfaat1, que codifica uma proteína transportadora de aminoácidos e está envolvido na resistência à cloroquina. O objectivo primordial deste trabalho centrou-se na caracterização dos genes acima descritos e na avaliação do seu possível papel em mecanismos de quimio-resistência em populações parasitárias provenientes de três regiões endémicas: Ruanda, República Democrática de São Tomé e Príncipe (RDSTP) e Brasil. Identificaram-se e caracterizaram-se os polimorfismos existentes em ambos os genes. O gene pfaat1 mostrou ser um gene altamente conservado e não revelou ter qualquer relação com a resistência às 4-aminoquinolinas. Pelo contrário, o gene pfcmu revelou possuir uma maior diversidade genética, tendo-se identificado nove mutações pontuais e três inserções, no conjunto de todos os isolados estudados. As amostras analisadas permitiram constituir catorze haplótipos, cuja diversidade demonstrou ser mais elevada nos países Africanos em comparação com o Brasil (Hd = 0,964 ± 0,077, 0,750 ± 0,139 e 0,250 ± 0,180 no Ruanda, RDSTP e Brasil, respectivamente). Alguns dos polimorfismos identificados revelaram especificidade geográfica. Identificou-se uma associação significativa entre uma mutação no gene pfcmu (G479A) e a susceptibilidade in vitro à dihidroartemisinina, em isolados provenientes do Ruanda (p = 0.0207). Estes resultados sugerem que polimorfismos no gene pfcmu podem estar envolvidos nos mecanismos de resistência do parasita P. falciparum aos derivados da artemisinina. Por conseguinte, estudos futuros envolvendo este gene e as respostas aos derivados da artemisinina, revestem-se de especial importância no contexto actual de uma vigilância eficaz da resistência a estes compostos

Influência da Espiritualidade no Idoso

O presente estudo tem como finalidade estudar a influência da espiritualidade na vida do idoso. Os seus objectivos são: conhecer as características sociodemográficas, avaliar o nível de espiritualidade e relacionar o nível de espiritualidade com algumas variáveis sociodemográficas num grupo de 101 idosos, 25 do sexo masculino (24,8%) e 76 do sexo feminino (75,2%), com idades compreendidas entre os 65 e os 95 anos de idade que frequentam centros de dia e lares do distrito de Leiria. Este estudo é transversal, de cariz quantitativo e do tipo descritivo e correlacional. O instrumento utilizado na recolha de dados foi um Questionário Sociodemográfico e a “Escala de Avaliação da Espiritualidade”, elaborada por Cândida Pinto e José Luís Pais-Ribeiro. Os idosos participantes neste estudo têm uma forte crença em Deus e atribuem uma grande importância à religião. O nível de espiritualidade encontrado na amostra é baixo. Relativamente aos dois factores analisados na Escala de Avaliação da Espiritualidade (Factor 1: “Crenças” e Factor 2: “Esperança/Optimismo”), o factor “Crenças” é o predominante na espiritualidade dos idosos em estudo. Não há relação estatisticamente significativa entre o nível de espiritualidade e a idade, contacto com familiares, existência de doença crónica e percepção de saúde. Contudo, o estudo sugere uma relação entre o nível de espiritualidade e a crença em Deus, grau de religiosidade, importância dada à religião, percepção de qualidade de vida, percepção de satisfação com a vida e percepção de felicidade. / This essay aims to study the influence of spirituality in the elderly’s life. Its objectives are: to identify the sociodemographic characteristics, to evaluate the level of spirituality and to relate the spiritual level with some of the sociodemographic variables in a group of 101 elderly, being 25 male (24,8%) and 76 female (75,2%), aged between 65 and 95 years old and attending day care and nursing homes in the district of Leiria. The present study is transversal, quantitative, descriptive and correlational. To gather the data, it was applied a sociodemographic questionnaire using the “Spirituality Evaluation Scale”, drawn up by Cândida Pinto and José Luís Pais-Ribeiro. The elderly that participated in this study have a deep belief in God and give a great importance to religion. The level of spirituality found in the sample is low. Regarding the two coefficients analysed in the Spirituality Evaluation Scale (Coefficient 1: “Beliefs” and Coefficient 2: “Hope/Optimism”), the coefficient “Beliefs” prevails in the spirituality of the studied elderly. There is no statistically significant relationship between the spirituality level and the age, contact with relatives, chronic disease and health perception. Yet, the study suggests a relationship between the level of spirituality and the belief in God, level of religiousness, religion significance and perceptions regarding quality of life, life satisfaction and happiness

Lack of K13 mutations in Plasmodium falciparum persisting after artemisinin combination therapy treatment of Kenyan children.

BACKGROUND: Studies in Southeast Asia reported a strong relationship between polymorphisms at the propeller domain of the Kelch 13 (K13) protein encoded by the Plasmodium falciparum k13 (pfk13) gene and delayed parasite clearance after artemisinin treatment. In Africa, P. falciparum remains susceptible and combination therapy regimens which include an artemisinin component display good efficacy. Using quantitative real-time PCR (qPCR), sub-microscopic persistence of P. falciparum has previously been reported in one-third of children treated with artemisinin combination therapy (ACT) in western Kenya. In this study, further investigation was made to evaluate whether these sub-microscopic residual parasites also harbour mutations at the propeller region of pfk13 and whether the mutations, if any, affect treatment outcome. METHODS: The pfk13 propeller domain was genotyped in DNA samples obtained in 2009 from Kenyan children treated with artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DP). Paired samples at pre-treatment (day 0) and day of treatment failure (day 28 or 42) for 32 patients with documented recurrent parasitaemia were available for genotyping. Additional day 3 DNA samples were available for 10 patients. RESULTS: No mutation associated with artemisinin resistance in Southeast Asia was observed. Only one DP-treated patient harboured a non-synonymous mutation at codon 578 (A578S) of pfk13-propeller gene in the day 0 sample, but this allele was replaced by the wild-type (A578) form on day 3 and on the day of recurrent parasitaemia. The mutation at amino acid codon 578 showed no association with any phenotype. Polymorphisms in pfk13 were not responsible for parasite persistence and gametocyte carriage in the children treated with ACT. CONCLUSION: This study contributes to the ongoing surveillance of suspected artemisinin resistance parasites in Africa by providing baseline prevalence of k13-propeller mutations in western Kenya with samples collected from a longitudinal study. Clinical Trials Registration NCT00868465

Culture-adapted Plasmodium falciparum isolates from UK travellers: in vitro drug sensitivity, clonality and drug resistance markers.

BACKGROUND: The screening of lead compounds against in vitro parasite cultures is an essential step in the development of novel anti-malarial drugs, but currently relies on laboratory parasite lines established in vitro during the last century. This study sought to establish in continuous culture a series of recent Plasmodium falciparum isolates to represent the current parasite populations in Africa, all of which are now exposed to artemisinin combination therapy. METHODS: Pre-treatment P. falciparum isolates were obtained in EDTA, and placed into continuous culture after sampling of DNA. One post-treatment blood sample was also collected for each donor to monitor parasite clonality during clearance in vivo. IC₅₀ estimates were obtained for 11 anti-malarial compounds for each established parasite line, clonal multiplicity measured in vivo and in vitro, and polymorphic sites implicated in parasite sensitivity to drugs were investigated at the pfmdr1, pfcrt, pfdhfr, pfdhps and pfap2mu loci before and after treatment, and in the cultured lines. RESULTS: Plasmodium falciparum isolates from seven malaria patients with recent travel to three West African and two East African countries were successfully established in long-term culture. One of these, HL1211, was from a patient with recrudescent parasitaemia 14 days after a full course of artemether-lumefantrine. All established culture lines were shown to be polyclonal, reflecting the in vivo isolates from which they were derived, and at least two lines reliably produce gametocytes in vitro. Two lines displayed high chloroquine IC₅₀ estimates, and carried the CVIET haplotype at codons 72-76, whereas the remaining five lines carried the CVMNK haplotype and were sensitive in vitro. All were sensitive to the endoperoxides dihydroartemisinin and OZ277, but IC₅₀ estimates for lumefantrine varied, with the least sensitive parasites carrying pfmdr1 alleles encoding Asn at codon 86. CONCLUSIONS: This study describes the establishment in continuous culture, in vitro drug sensitivity testing and molecular characterization of a series of multiclonal P. falciparum isolates taken directly from UK malaria patients following recent travel to various malaria-endemic countries in Africa. These "HL" isolates are available as an open resource for studies of drug response, antigenic diversity and other aspects of parasite biology

Estrutura e diversidade genética de oliveiras centenárias da região Transmontana

As oliveiras são componentes importantes da paisagem Transmontana, região onde ainda é possível encontrar árvores centenárias. Estas, podem ser uma fonte de produtos diferenciados azeitonas de mesa e azeites estratégia que interessa valorizar. Contudo, o desconhecimento sobre a sua diversidade e estrutura populacional na região poderá inviabilizar esta estratégia, por não ser possível definir medidas de gestão e conservação adequadas para a sua proteção. Neste sentido, no presente trabalho pretendeu-se caracterizar geneticamente, através da análise por microssatélites, 17 exemplares de oliveiras centenárias da região de Trás-os-Montes e elucidar possíveis relações de filogenia com 15 exemplares de cultivares conhecidas na região.info:eu-repo/semantics/publishedVersio

The Mu subunit of Plasmodium falciparum clathrin-associated adaptor protein 2 modulates in vitro parasite response to artemisinin and quinine.

The emergence of drug-resistant parasites is a serious threat faced by malaria control programs. Understanding the genetic basis of resistance is critical to the success of treatment and intervention strategies. A novel locus associated with antimalarial resistance, ap2-mu (encoding the mu chain of the adaptor protein 2 [AP2] complex), was recently identified in studies on the rodent malaria parasite Plasmodium chabaudi (pcap2-mu). Furthermore, analysis in Kenyan malaria patients of polymorphisms in the Plasmodium falciparum ap2-mu homologue, pfap2-mu, found evidence that differences in the amino acid encoded by codon 160 are associated with enhanced parasite survival in vivo following combination treatments which included artemisinin derivatives. Here, we characterize the role of pfap2-mu in mediating the in vitro antimalarial drug response of P. falciparum by generating transgenic parasites constitutively expressing codon 160 encoding either the wild-type Ser (Ser160) or the Asn mutant (160Asn) form of pfap2-mu. Transgenic parasites carrying the pfap2-mu 160Asn allele were significantly less sensitive to dihydroartemisinin using a standard 48-h in vitro test, providing direct evidence of an altered parasite response to artemisinin. Our data also provide evidence that pfap2-mu variants can modulate parasite sensitivity to quinine. No evidence was found that pfap2-mu variants contribute to the slow-clearance phenotype exhibited by P. falciparum in Cambodian patients treated with artesunate monotherapy. These findings provide compelling evidence that pfap2-mu can modulate P. falciparum responses to multiple drugs. We propose that this gene should be evaluated further as a potential molecular marker of antimalarial resistance

Influence of the different “patient global assessment” formulations on disease activity score by different indices in rheumatoid arthritis

© 2018, International League of Associations for Rheumatology (ILAR). Patient global assessment (PGA) is included in almost all rheumatoid arthritis (RA) composite disease activity indices and definitions of remission. However, different PGA formulations exist and are used interchangeably in research and clinical practice. We investigated how five different PGA formulations used in four disease indices affect the remission rates. This was an ancillary analysis of data from a cross-sectional study in patients with RA. The data comprised the following: 28-joint counts, C-reactive protein, and five PGA formulations. Remission rate variation was assessed using five PGA formulations in each index (ACR/EULAR Boolean, CDAI, SDAI, and DAS28-CRP). PGA agreement was assessed by the following: Pearson’s correlation; Bland-Altman plots; paired samples t test; and establishing the proportion of patients who scored (i) all formulations within an interval of 20mm and (ii) each formulation ≤ 10mm. This analysis included 191 patients. PGA formulations presented good correlations (≥ 0.65), but Bland-Altman plots showed clinically significant differences, which were statistically confirmed by comparison of means. Just over a half (51.8%) of patients scored all PGA formulations within a 20-mm interval. The proportion of those scoring ≤ 10mm varied from 11.5 to 16.2%. When different formulations of PGA were used in each index, remission differences of up to 4.7, 4.7, 6.3, and 5.2% were observed. When formulations were used in their respective indices, as validated, the remission rates were similar (13.1, 13.6, 14.1, and 18.3%). Using PGA formulations interchangeably may have implications in the assessment of disease activity and in the attainment of remission, and this can impact upon management decisions

Failure of rapid diagnostic tests in Plasmodium falciparum malaria cases among travelers to the UK and Ireland: Identification and characterisation of the parasites.

OBJECTIVES: Our objective was to systematically investigate false-negative histidine-rich protein 2 rapid diagnostic tests (HRP2-RDT) in imported Plasmodium falciparum malaria cases from travelers to the UK and the Republic of Ireland (RoI). METHODS: Five imported malaria cases in travellers returning to the UK and RoI from East Africa were reported to the PHE Malaria Reference Laboratory as negative according to histidine-rich protein (HRP2)-RDT. The cases were systematically investigated using microscopic, RDT, molecular, genomic, and in in vitro approaches. RESULTS: In each case, HRP2-RDT was negative, whereas microscopy confirmed the presence of P. falciparum. Further analysis revealed that the genes encoding HRP2 and HRP3 were deleted in three of the five cases. Whole-genome sequencing in one of these isolates confirmed deletions in P. falciparum chromosomes 8 and 13. Our study produced evidence that the fourth case, which had high parasitemia at clinical presentation, was a rare example of antigen saturation ('prozone-like effect'), leading to a false negative in the HRP2-RDT, while the fifth case was due to low parasitemia. CONCLUSIONS: False-negative HRP2-RDT results with P. falciparum are concerning. Our findings emphasise the necessity of supporting the interpretation of RDT results with microscopy, in conjunction with clinical observations, and sets out a systematic approach to identifying parasites carrying pfhrp2 and pfhrp3 deletions