Luiz Hildebrando Pereira da Silva

Asymptomatic Carriers of Plasmodium spp. as Infection Source for Malaria Vector Mosquitoes in the Brazilian Amazon

Abstract We have described the existence of asymptomatic carriers of Plasmodium vivax and Plasmodium falciparum infections in native Amazon populations. Most of them had low parasitemias, detected only by polymerase chain reaction (PCR). Because they remain symptomless and untreated, we wanted to determine whether they could infect Anopheles darlingi Root, the main Brazilian vector, and act as disease reservoirs. Fifteen adult asymptomatic patients (PCR positive only) were selected, and experimental infections of mosquitoes were performed by direct feeding and by a membrane-feeding system. Seventeen adult symptomatic patients with high parasitemias were used as controls. We found an infection rate in An. darlingi of 1.2% for the asymptomatic carriers and 22% for the symptomatic carriers. Although the asymptomatic group infected mosquitoes at a much lower rate, these patients remain infective longer than treated, symptomatic patients. Also, the prevalence of asymptomatic infections is 4 to 5 times higher than symptomatic infections among natives. These results have implications for the malaria control program in Brazil, which focuses essentially on the treatment of symptomatic patients.

Malaria primes the innate immune response due to interferon-gamma induced enhancement of toll-like receptor expression and function

Malaria-induced sepsis is associated with an intense proinflammatory cytokinemia for which the underlying mechanisms are poorly understood. It has been demonstrated that experimental infection of humans with Plasmodium falciparum primes Toll-like receptor (TLR)-mediated proinflammatory responses. Nevertheless, the relevance of this phenomenon during natural infection and, more importantly, the mechanisms by which malaria mediates TLR hyperresponsiveness are unclear. Here we show that TLR responses are boosted in febrile patients during natural infection with P. falciparum. Microarray analyses demonstrated that an extraordinary percentage of the up-regulated genes, including genes involving TLR signaling, had sites for IFN-inducible transcription factors. To further define the mechanism involved in malaria-mediated “priming,” we infected mice with Plasmodium chabaudi. The human data were remarkably predictive of what we observed in the rodent malaria model. Malaria-induced priming of TLR responses correlated with increased expression of TLR mRNA in a TLR9-, MyD88-, and IFNγ-dependent manner. Acutely infected WT mice were highly susceptible to LPS-induced lethality while TLR9−/−, IL12−/− and to a greater extent, IFNγ−/− mice were protected. Our data provide unprecedented evidence that TLR9 and MyD88 are essential to initiate IL12 and IFNγ responses and favor host hyperresponsiveness to TLR agonists resulting in overproduction of proinflammatory cytokines and the sepsis-like symptoms of acute malaria.

The rise and fall of malaria in a west African rural community, Dielmo, Senegal, from 1990 to 2012: a 22 year longitudinal study

BACKGROUND A better understanding of the effect of malaria control interventions on vector and parasite populations, acquired immunity, and burden of the disease is needed to guide strategies to eliminate malaria from highly endemic areas. We monitored and analysed the changes in malaria epidemiology in a village community in Senegal, west Africa, over 22 years. METHODS Between 1990 and 2012, we did a prospective longitudinal study of the inhabitants of Dielmo, Senegal, to identify all episodes of fever and investigate the relation between malaria host, vector, and parasite. Our study included daily medical surveillance with systematic parasite detection in individuals with fever. We measured parasite prevalence four times a year with cross-sectional surveys. We monitored malaria transmission monthly with night collection of mosquitoes. Malaria treatment changed over the years, from quinine (1990-94), to chloroquine (1995-2003), amodiaquine plus sulfadoxine-pyrimethamine (2003-06), and finally artesunate plus amodiaquine (2006-12). Insecticide-treated nets (ITNs) were introduced in 2008. FINDINGS We monitored 776 villagers aged 0-101 years for 2 378 150 person-days of follow-up. Entomological inoculation rate ranged from 142·5 infected bites per person per year in 1990 to 482·6 in 2000, and 7·6 in 2012. Parasite prevalence in children declined from 87% in 1990 to 0·3 % in 2012. In adults, it declined from 58% to 0·3%. We recorded 23 546 fever episodes during the study, including 8243 clinical attacks caused by Plasmodium falciparum, 290 by Plasmodium malariae, and 219 by Plasmodium ovale. Three deaths were directly attributable to malaria, and two to severe adverse events of antimalarial drugs. The incidence of malaria attacks ranged from 1·50 attacks per person-year in 1990 to 2·63 in 2000, and to only 0·046 in 2012. The greatest changes were associated with the replacement of chloroquine and the introduction of ITNs. INTERPRETATION Malaria control policies combining prompt treatment of clinical attacks and deployment of ITNs can nearly eliminate parasite carriage and greatly reduce the burden of malaria in populations exposed to intense perennial malaria transmission. The choice of drugs seems crucial. Rapid decline of clinical immunity allows rapid detection and treatment of novel infections and thus has a key role in sustaining effectiveness of combining artemisinin-based combination therapy and ITNs despite increasing pyrethroid resistance. FUNDING Pasteur Institutes of Dakar and Paris, Institut de Recherche pour le Développement, and French Ministry of Cooperation.

THE USE OF BIODIVERSITY AS SOURCE OF NEW CHEMICAL ENTITIES AGAINST DEFINED MOLECULAR TARGETS FOR TREATMENT OF MALARIA, TUBERCULOSIS, AND T-CELL MEDIATED DISEASES – A REVIEW

The modern approach to the development of new chemical entities against complex diseases, especially the neglected endemic diseases such as tuberculosis and malaria, is based on the use of defined molecular targets. Among the advantages, this approach allows (i) the search and identification of lead compounds with defined molecular mechanisms against a defined target (e.g. enzymes from defined pathways), (ii) the analysis of a great number of compounds with a favorable cost/benefit ratio, (iii) the development even in the initial stages of compounds with selective toxicity (the fundamental principle of chemotherapy), (iv) the evaluation of plant extracts as well as of pure substances. The current use of such technology, unfortunately, is concentrated in developed countries, especially in the big pharma. This fact contributes in a significant way to hamper the development of innovative new compounds to treat neglected diseases. The large biodiversity within the territory of Brazil puts the country in a strategic position to develop the rational and sustained exploration of new metabolites of therapeutic value. The extension of the country covers a wide range of climates, soil types, and altitudes, providing a unique set of selective pressures for the adaptation of plant life in these scenarios. Chemical diversity is also driven by these forces, in an attempt to best fit the plant communities to the particular abiotic stresses, fauna, and microbes that co-exist with them. Certain areas of vegetation (Amazonian Forest, Atlantic Forest, Araucaria Forest, Cerrado-Brazilian Savanna, and Caatinga) are rich in species and types of environments to be used to search for natural compounds active against tuberculosis, malaria, and chronic-degenerative diseases. The present review describes some strategies to search for natural compounds, whose choice can be based on ethnobotanical and chemotaxonomical studies, and screen for their ability to bind to immobilized drug targets and to inhibit their activities. Molecular cloning, gene knockout, protein expression and purification, N-terminal sequencing, and mass spectrometry are the methods of choice to provide homogeneous drug targets for immobilization by optimized chemical reactions. Plant extract preparations, fractionation of promising plant extracts, propagation protocols and definition of in planta studies to maximize product yield of plant species producing active compounds have to be performed to provide a continuing supply of bioactive materials. Chemical characterization of natural compounds, determination of mode of action by kinetics and other spectroscopic methods (MS, X-ray, NMR), as well as in vitro and in vivo biological assays, chemical derivatization, and structure-activity relationships have to be carried out to provide a thorough knowledge on which to base the search for natural compounds or their derivatives with biological activity.

Cross-reactive antigenic determinants present on different Plasmodium falciparum blood-stage antigens.

Summary A gene encoding a previously undescribed antigen of Plasmodium falciparum has been isolated from a genomic expression library by use of a pool of human immune sera. Northern blot analysis indicated that the gene is expressed at the late stages of the intra–erythrocytic cycle. This antigen, 332, contains a series of degenerated amino acid repeats. Human antibodies affinity–purified on the 332 recombinant antigen reacted with a family of parasite proteins that are products of different genes. We identified antigens 11.1 and Pf 155–RESA as members of this family and confirmed, using a human monoclonal antibody, the presence of cross–reacting determinants. The sequences of these antigens also share some structural homologies. The significance of this family of blood–stage antigens is discussed.

In vivo time course of synthesis and processing of major schizont membrane polypeptides in Plasmodium falciparum

A rapid method of separating membrane co-sedimentable and soluble components of Plasmodium infected erythrocytes is presented. We propose a nomenclature for major P. falciparum polypeptides, applicable to different isolates and based on their cellular location and stage specificity. For four of these polypeptides (185 kDa = Mp1; 120 kDa = Mp3; 76 kDa = Mp5; 90 kDa= Sp2) supposed to play a role in protective immunity, monospecific antibodies were available. We have studied their fate at the time of merozoïte release and reinvasion, and the possible correlations between these polypeptides, by pulse-chase experiments.

Symptomless Plasmodium vivax infections in native Amazonians

Naturally acquired immunity to falciparum malaria has been recorded in holoendemic and hyperendemic Africa. , 2 Similar records do not exist for vivax malaria. Although individuals repeatedly infected with Plasmodium vivax m a y have attenuated symptoms, absence of symptoms has never been described in endemic areas of vivax malaria. , 3 W e provide evidence of symptomless vivax malaria in a riverine Amazonian population in the state of Rondonia, Brazil. Colonisers who came from malaria-free southern Brazil between 1960 and 1980 and their immediate descendants constitute more than 95% of the Rondonian population of 1 2 00 000 inhabitants. When those migrants arrived, they encountered a small native population that had been living for generations in settlements scattered along rivers. From the 1960s onwards, malaria spread to immigrants. It was often severe but remained endemic and less severe in the native people. The presence of blood parasites, either P falciparum or P vivax, was always accompanied by symptoms in migrants. In the riverine population a few people had blood parasites in absence of symptoms. W e considered that some of the native population might be immune to malaria and also serve as reservoirs of the d i s e a s e . To test this hypothesis we studied a native population, adding PCR-amplification of ribosomal DNA to clinical examination and blood-smear microscopy for the diagnosis of vivax malaria. After giving informed consent, 183 inhabitants (92% of a village population) were followed up from September, 1998. 14 cases of vivax malaria were diagnosed up to January, 1999. All cases had malaria symptoms with positive blood smears and positive PCR. The majority of the patients (82%) had lived for more than 1 year in the village and were less than 16 years old. We also detected 25 individuals (68% of them older than 16 years) who were PCR-positive for P vivax, but without symptoms. 16 were put under medical supervision for 30 days. Eight had positive blood smears with very low

Plaque antibody selection: rapid immunological analysis of a large number of recombinant phage clones positive to sera raised against Plasmodium falciparum antigens

A library of Plasmodium falciparum genomic DNA on the lambda gt11 phage vector was screened for clones positive to a rabbit serum raised against a purified fraction of P. falciparum proteins and a pool of sera from malaria patients. The positive clones were characterized with antibodies purified by the plaque antibody selection technique. This technique consist of purifying specific antibodies on a nitrocellulose filter blotted directly on a lawn of plaques of an antigen-producing phage clone. The purified antibodies are then used as a probe in a Western blot of parasite protein extract, for preliminary characterization of the clones. Using this method, two different clones coding for P. falciparum antigens were identified with the rabbit serum and about 20 with the human sera. This method can be of general use, i.e. it is not limited to parasite systems, and facilitates the immunological analysis and identification of a large number of clones.

A Reduced Risk of Infection with Plasmodium vivax and Clinical Protection against Malaria Are Associated with Antibodies against the N Terminus but Not the C Terminus of Merozoite Surface Protein 1

ABSTRACT Progress towards the development of a malaria vaccine against Plasmodium vivax, the most widely distributed human malaria parasite, will require a better understanding of the immune responses that confer clinical protection to patients in regions where malaria is endemic. The occurrence of clinical protection in P. vivax malaria in Brazil was first reported among residents of the riverine community of Portuchuelo, in Rondônia, western Amazon. We thus analyzed immune sera from this same human population to determine if naturally acquired humoral immune responses against the merozoite surface protein 1 of P. vivax, PvMSP1, could be associated with reduced risk of infection and/or clinical protection. Our results demonstrated that this association could be established with anti-PvMSP1 antibodies predominantly of the immunoglobulin G3 subclass directed against the N terminus but not against the C terminus, in spite of the latter being more immunogenic and capable of natural boosting. This is the first report of a prospective study of P. vivax malaria demonstrating an association of reduced risk of infection and clinical protection with antibodies against an antigen of this parasite.

Urban and suburban malaria in Rondônia (Brazilian Western Amazon) II: perennial transmissions with high anopheline densities are associated with human environmental changes

Longitudinal entomological surveys were performed in Vila Candelária and adjacent rural locality of Bate Estaca concomitantly with a clinical epidemiologic malaria survey. Vila Candelária is a riverside periurban neighborhood of Porto Velho, capital of the state of Rondônia in the Brazilian Amazon. High anopheline densities were found accompanying the peak of rainfall, as reported in rural areas of the region. Moreover, several minor peaks of anophelines were recorded between the end of the dry season and the beginning of the next rainy season. These secondary peaks were related to permanent anopheline breeding sites resulting from human activities. Malaria transmission is, therefore, observed all over the year. In Vila Candelária, the risk of malaria infection both indoors and outdoors was calculated as being 2 and 10/infecting bites per year per inhabitant respectively. Urban malaria in riverside areas was associated with two factors: (1) high prevalence of asymptomatic carriers in a stable human population and (2) high anopheline densities related to human environmental changes. This association is probably found in other Amazonian urban and suburban communities. The implementation of control measures should include environmental sanitation and better characterization of the role of asymptomatic carriers in malaria transmission.