Transglutaminases do mosquito Anopheles gambiae: Caracterização genética, bioquímica e funcional durante a infecção por Plasmodium berghei

A malária constitui um problema de saúde pública, que tem vindo a agravar-se, sendo crescente a necessidade de estratégias renovadas para o seu controlo, como a interrupção do ciclo esporogónico. Deste modo, é essencial compreender as respostas imunológicas de Anopheles anti-Plasmodium. Demonstrou-se anteriormente, que a inibição de transglutaminases, enzimas que participam em vários processos biológicos ao catalisarem a formação de ligações covalentes entre péptidos, agrava a infecção em mosquitos pelo parasita. O presente trabalho tem por objectivo caracterizar as transglutaminases AGAP009098 e AGAP009100 de Anopheles gambiae. Os métodos utilizados para este efeito foram: a sequenciação de regiões dos genes AGAP009098 e AGAP009100; a clonagem molecular de fragmentos da região codificante do gene AGAP009098, usando o vector plasmídico pET–28a(+) e Escherichia coli como sistema de expressão; e PCR em Tempo Real para analisar a expressão relativa dos genes AGAP009098 e AGAP009100 nos diferentes os estádios de desenvolvimento. AGAP009098 é expressa ubiquamente e AGAP009100 a partir do estádio pupa. Estes resultados apontam para a conclusão de que AGAP009098 e AGAP009100 poderão desempenhar funções em processos biológicos relevantes, por exemplo na defesa imunitária, ou no desenvolvimento. Os péptidos recombinantes, obtidos a partir da clonagem com sucesso de fragmentos da região codificante do gene AGAP009098, constituem uma ferramenta importante para averiguar a função destas TGases, no futuro.

In vivo and in vitro Plasmodium falciparum Resistance to Chloroquine, Amodiaquine and Quinine in the Brazilian Amazon

In order to study the chemoresistance of Plasmodium falciparum to commonly used antimalarial drugs in Brazil the authors have studied ten patients with falciparum malaria, acquired in the Brazilian Amazon region. Patients were submitted to in vivo study of drug sensitivity, after chemotherapy with either 4-aminoquinolines (chloroquine or amodiaquine) or quinine. Adequate drug absorption was confirmed by standard urine excretion tests for antimalarials. Eight patients could be followed up to 28 days. Among these in vivo resistance (R I and R II responses) was seen in all patients who received 4-amino-quinolines. One patient treated with quinine exhibited a R III response. Peripheral blood samples of the same patients were submitted to in vitro microtests for sensitivity to antimalarials. Out of nine successful tests, resistance to chloroquine and amodiaquine was found in 100% and resistance to quinine in 11.11% of isolates. Probit analysis of log dose-response was used to determine effective concentrations EC50, EC90 and EC99 to the studied drugs. Good correlation between in vivo and in vitro results was seen in six patients. The results emphasize high levels of P. falciparum resistance to 4- aminoquinolines and suggest an increase in resistance to quinine in the Brazilian Amazon region, reinforcing the need for continuous monitoring of drug sensitivity to adequate chemotherapy according to the most efficacious drug regimens

STANDARDIZATION OF PROCEDURES OF Plasmodium falciparum ANTIGEN PREPARATION FOR SEROLOGIC TESTS

The objective of the present study is to standardize the technical variables for preparation and storage of Plasmodium falciparum and of antigen components extracted with the amphoteric detergent Zwittergent. P. falciparum obtained from in vitro culture was stored at different temperatures and for different periods of time. For each variable, antigen components of the parasite were extracted in the presence or absence of protease inhibitors and submitted or not to later dialysis. Products were stored for 15, 30 and 60 days at different temperatures and immunological activity of each extract was determined by SDS-PAGE and ELISA using positive or negative standard sera for the presence of IgG directed to blood stage antigens of P. falciparum. Antigen extracts obtained from parasites stored at -20oC up to 10 days or at -70oC for 2 months presented the best results, showing well-defined bands on SDS-PAGE and Western blots and presenting absorbance values in ELISA that permitted safe differentiation between positive and negative sera.

Au-nanossondas aplicação na detecção de Mycobacterium tuberculosis e plasmodium berghei

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para a obtenção do grau de Mestre em Biotecnologia

In vitro evaluation of quinidine sensitivity in brazilian Plasmodium falciparum isolates: comparative analysis to quinine and chloroquine

Falciparum malaria represents a serious and an increasing world public health problem due to the acquired parasite's resistance to the most available drugs. In some endemic areas, quinidine, a diastereoisomer of the antimalarial quinine, has been employed for replacing the latter. In order to evaluate the use of quinidine as an alternative to the increasing loss of quinine effectiveness in Brazilian P. falciparum strains, as has been observed in the Amazon area, we have assayed quinidine, quinine and chloroquine. The in vitro microtechnique was employed. All isolates showed to be highly resistant to chloroquine. Resistance to quinine was not noted although high MIC (minimal inhibitory concentration) values have been observed. These data corroborate the decreasing sensitivity to quinine in strains from Brazil. Quinidine showed IC50 from 0.053 to 4.577 mumol/L of blood while IC50 from 0.053 to 8.132 mumol/L of blood was estimated for quinine. Moreover, clearance of the parasitemia was observed in concentrations lower than that used for quinidine in antiarrhythmic therapy, confirming our previous data. The results were similar to African isolate.

Evaluation of a rapid dipstick test, Malar-CheckTM, for the diagnosis of Plasmodium falciparum malaria in Brazil

The present study was carried out to evaluate the Malar-CheckTM Pf test, an immunochromatographic assay that detects Plasmodium falciparum Histidine Rich Protein II, does not require equipment, and is easy and rapid to perform. In dilution assays performed to test sensitivity against known parasite density, Malar-CheckTMwere compared with thick blood smear (TBS), the gold standard for diagnosis. Palo Alto isolate or P. falciparum blood from patients with different parasitemias was used. The average cut-off points for each technique in three independent experiments were 12 and 71 parasites/mm³ (TBS and Malar-CheckTM, respectively). In the field assays, samples were collected from patients with fever who visited endemic regions. Compared to TBS, Malar-CheckTMyielded true-positive results in 38 patients, false-positive results in 3, true-negative results in 23, and false-negative result in 1. Malar-CheckTMperformed with samples from falciparum-infected patients after treatment showed persistence of antigen up to 30 days. Malar-CheckTM should aid the diagnosis of P. falciparum in remote areas and improve routine diagnosis even when microscopy is available. Previous P. falciparum infection, which can determine a false-positive test in cured individuals, should be considered. The prompt results obtained with the Malar-CheckTM for early diagnosis could avoid disease evolution to severe cases.

Clinical and laboratory findings of Plasmodium vivax malaria in Colombia, 2001

A descriptive study was carried out in 104 patients with Plasmodium vivax malaria, from the region of Turbo (Antioquia, Colombia). Clinical features and levels of hemoglobin, glycemia, serum bilirubin, alanine-aminotransferase (ALT), aspartate-aminotransferase (AST), creatinine and complete blood cell profile were established. 65% of the studied individuals were men and their mean age was 23. Of all individuals 59% had lived in the region for > 1 year and 91% were resident in the rural area. 42% were farmers and 35% had a history of malaria. The mean parasitaemia was 5865 parasites/mm³. The evolution of the disease was short (average of 4.0 days). Fever, headache and chills were observed simultaneously in 91% of the cases while the most frequent signs were palmar pallor (46%), jaundice (15%), hepatomegaly (17%), and spleen enlargement (12%). Anemia was found in 39% of the women and in 51% of the men, 8% of individuals had thrombocytopaenia and 41% had hypoglycemia.

Assessment of chloroquine single dose treatment of malaria due to Plasmodium vivax in Brazilian Amazon

Malaria regions of the Amazon basin have been characterized by difficult access and non-compliance of the patients to treatment. In an attempt to assess the schizonticide efficacy of chloroquine in a single dose of 600 mg, the authors realized a double-blind, placebo-controlled trial in 132 outpatients with vivax malaria. Patients were distributed into two groups: group CPLA, given chloroquine 600 mg (single dose) on the first day of treatment, and two doses of placebo on second and third days. Group CHLO, given chloroquine 600 mg on first day and 450 mg on second and third day. Geometric means of the parasite density during the follow-up was similar in both groups. No differences were observed in the parasitological cure between the two groups (p = 0.442). There was clinical and parasitological efficacy in treatment of patients given a single-dose of chloroquine. This suggests that its restricted use could be indicated in remote areas of Brazilian Amazon Region, nevertheless the inadequate response of three patients indicates the need for further studies.

Rapid turnover of Plasmodium falciparum var gene transcripts and genotypes during natural non-symptomatic infections

The var genes of Plasmodium falciparum code for the antigenically variant erythrocyte membrane proteins 1 (PfEMP1), a major factor for cytoadherence and immune escape of the parasite. Herein, we analyzed the var gene transcript turnover in two ongoing, non-symptomatic infections at sequential time points during two weeks. The number of different circulating genomes was estimated by microsatellite analyses. In both infections, we observed a rapid turnover of plasmodial genotypes and var transcripts. The rapidly changing repertoire of var transcripts could have been caused either by swift elimination of circulating var-transcribing parasites stemming from different or identical genetic backgrounds, or by accelerated switching of var gene transcription itself.

Susceptibility of two karyotypic forms of Anopheles aconitus (Diptera: Culicidae) to Plasmodium falciparum and P. vivax

Four laboratory-raised colonies of two karyotypic forms of Anopheles aconitus, i.e., Form B (Chiang Mai and Phet Buri strains) and C (Chiang Mai and Mae Hong Son strains), were experimentally infected with Plasmodium falciparum and P. vivax using an artificial membrane feeding technique and dissected eight and 12 days after feeding for oocyst and sporozoite rates, respectively. The results revealed that An. aconitus Form B and C were susceptible to P. falciparum and P. vivax, i.e., Form B (Chiang Mai and Phet Buri strains/P. falciparum and P. vivax) and Form C (Chiang Mai and Mae Hong Son strains/P. vivax). Comparative statistical analyses of the oocyst rates, average number of oocysts per infected midgut and sporozoite rates among all strains of An. aconitus Form B and C to the ingroup control vectors, An. minimus A and C, exhibited mostly no significant differences, confirming the high potential vector of the two Plasmodium species. The sporozoite-like crystals found in the median lobe of the salivary glands, which could be a misleading factor in the identification of true sporozoites in salivary glands were found in both An. aconitus Form B and C.

The role of detoxification in the mosquito Anopheles gambiae response to Plasmodium infection

Rute C. Félix PALAVRAS-CHAVE: Malária, mosquito vector, Anopheles gambiae, parasita, Plasmodium berghei, infecção, enzimas de detoxificação, citocromos P450, tubulinas A malária, uma das doenças mais devastadoras que ocorrem em África é causada por um parasita do género Plasmodium e é transmitida aos humanos por mosquitos vectores do género Anopheles durante a refeição de sangue. Apesar da resposta do mosquito à infecção por Plasmodium ter vindo a ser intensamente estudada nos últimos anos, as interacções entre o mosquito vector e o parasita são muito complexas e, estão longe de serem completamente compreendidas. Este estudo tem como objectivo principal contribuir para o conhecimento da resposta do mosquito à infecção por Plasmodium, focando-se no papel das enzimas de detoxificação. Para atingir este objectivo realizouse uma análise transcriptómica com microarrays, com o intuito de identificar alterações de transcrição de enzimas de detoxificação no mosquito Anopheles gambiae em resposta à infecção por Plasmodium. Esta análise permitiu identificar alterações na expressão de 254 genes de destoxificação no estômago e corpo gordo de A. gambiae durante a invasão do intestino médio pelos oocinetos e durante a libertação dos esporozoítos do oocisto. Os resultados mostraram que a invasão do intestino médio pelos oocinetos causou alterações num maior número de genes em ambos os tecidos estudados, sendo o intestino médio do mosquito o tecido mais afectado nas duas fases da infecção do parasita. De todos os genes de destoxificação com expressão alterada, as tubulinas e os citocromos P450 destacaram-se e foram escolhidos para continuar o estudo. As tubulinas foram seleccionadas porque estão associadas à invasão do epitélio do intestino médio e a sua função na resposta à invasão do Plasmodium ainda não está bem definida. Os citocromos P450 foram seleccionados porque já foram descritos como tendo a expressão alterada em resposta ao Plasmodium e a outras infecções. Para identificar e caracterizar o papel das tubulinas durante a infecção pelo parasita e a sua possível associação com os citocromos P450 foi utilizado o silenciamento génico por RNA de interferência e a injecção de inibidores químicos de tubulinas. O silenciamento e co-silenciamento das tubulinas causaram um aumento da taxa e intensidade da infecção. No entanto, apesar de o aumento ser consistente não foi significativo. Por outro lado, a injecção de paclitaxel, um inibidor de tubulinas, aumentou significativamente a taxa e intensidade da infecção, fortalecendo a hipótese do envolvimento das tubulinas na resposta à infecção por Plasmodium. Este trabalho também mostrou que o co-silenciamento da tubulina A e tubulina B e a injecção do inibidor de tubulinas colchicine causam alterações significativas na expressão da CYP6Z2, sendo este proposto como um possível elo de ligação entre as tubulinas e os citocromos P450. Finalmente, uma análise comparativa foi realizada para estudar as regiões promotoras dos citocromos P450: CYP6M2 e o CYP6Z1. Este estudo obteve novos dados sobre compostos que activam estes citocromos e quais os possíveis factores de transcrição envolvidos. Dos diferentes estímulos utilizados, a exposição a insecticidas e a bactérias foram os que mais afectaram estes citocromos. O conjunto total das diferentes abordagens utilizadas neste trabalho contribuiu para aumentar o conhecimento do papel das enzimas de destoxificação durante a passagem do parasita da malária pelo mosquito vector.

Sharing of antigens between Plasmodium falciparum and Anopheles albimanus

The presence of common antigens between Plasmodium falciparum and Anopheles albimanus was demonstrated. Different groups of rabbits were immunized with: crude extract from female An. albimanus (EAaF), red blood cells infected with Plasmodium falciparum (EPfs), and the SPf66 synthetic malaria vaccine. The rabbit's polyclonal antibodies were evaluated by ELISA, Multiple Antigen Blot Assay (MABA), and immunoblotting. All extracts were immunogenic in rabbits according to these three techniques, when they were evaluated against the homologous antigens. Ten molecules were identified in female mosquitoes and also in P. falciparum antigens by the autologous sera. The electrophoretic pattern by SDS-PAGE was different for the three antigens evaluated. Cross-reactions between An. albimanus and P. falciparum were found by ELISA, MABA, and immunoblotting. Anti-P. falciparum and anti-SPf66 antibodies recognized ten and five components in the EAaF crude extract, respectively. Likewise, immune sera against female An. albimanus identified four molecules in the P. falciparum extract antigen. As far as we know, this is the first work that demonstrates shared antigens between anophelines and malaria parasites. This finding could be useful for diagnosis, vaccines, and the study of physiology of the immune response to malaria.

Molecular screening of Plasmodium sp. asymptomatic carriers among transfusion centers from Brazilian Amazon region

The transmission of malaria in Brazil is heterogeneous throughout endemic areas and the presence of asymptomatic Plasmodium sp. carriers (APCs) in the Brazilian Amazon has already been demonstrated. Malaria screening in blood banks is based on the selection of donors in respect to possible risks associated with travel or residence, clinical evidence and/or inaccurate diagnostic methods thereby increasing the probability of transfusion-transmitted infection. We evaluated the frequency of APCs in four blood services in distinct areas of the Brazilian Amazon region. DNA was obtained from 400 human blood samples for testing using the phenol-chloroform method followed by a nested-PCR protocol with species-specific primers. The positivity rate varied from 1 to 3% of blood donors from the four areas with an average of 2.3%. All positive individuals had mixed infections for Plasmodium vivax and Plasmodium falciparum. No significant differences in the results were detected among these areas; the majority of cases originated from the transfusion centres of Porto Velho, Rondônia State and Macapá, Amapá State. Although it is still unclear whether APC individuals may act as reservoirs of the parasite, efficient screening of APCs and malaria patients in Brazilian blood services from endemic areas needs to be improved.

Cross sectional study reveals a high percentage of asymptomatic Plasmodium vivax infection in the Amazon Rio Negro area, Brazil

A parasitological, clinical, serological and molecular cross-sectional study carried out in a highly endemic malaria area of Rio Negro in the Amazon State, Brazil, revealed a high prevalence of asymptomatic Plasmodium vivax infection. A total of 109 persons from 25 families were studied in five villages. Ninety-nine inhabitants (90.8%) had at least one previous episode of malaria. Serology showed 85.7% and 46.9% of positivity when P. falciparum antigens and P. vivax MSP-1, respectively, were used. Twenty blood samples were PCR positive for P. vivax (20.4%) and no P. falciparum infection was evidenced by this technique. No individual presenting positive PCR reaction had clinical malaria during the survey neither in the six months before nor after, confirming that they were cases of asymptomatic infection. Only one 12 year old girl presented a positive thick blood smear for P. vivax. This is the first description of asymptomatic Plasmodium infection in this area studied.