Detection of Reticuloendotheliosis Virus in Muscovy Ducks, Wild Turkeys, and Chickens in Brazil.

Reticuloendotheliosis viruses (REVs) are known to cause immunosuppressive and oncogenic disease that affects numerous avian species. Reticuloendotheliosis viruses are present worldwide and recently have been reported in South America with cases of infected commercial flocks in Argentina. We surveyed for the presence of REV in birds from a state in the northern region of Brazil using real-time PCR. We report here the presence of REV in Brazil, detected in Muscovy Ducks (Cairina moschata), Wild Turkeys (Meleagris gallopavo), and chickens (Gallus gallus) at a relatively high prevalence (16.8%). Phylogenetic analysis indicated a close relationship of these strains to variants in the US. This study provides evidence of REV in the Amazon biome and provides a baseline for future surveillance of the virus in the region and throughout Brazil.

Recombinant proteins of Plasmodium malariae merozoite surface protein 1 (PmMSP1): Testing immunogenicity in the BALB/c model and potential use as diagnostic tool.

BACKGROUND: Plasmodium malariae is the third most prevalent human malaria-causing species and has a patchy, but ample distribution in the world. Humans can host the parasite for years without presenting significant symptoms, turning its diagnosis and control into a difficult task. Here, we investigated the immunogenicity of recombinant proteins of P. malariae MSP1. METHODS: Five regions of PmMSP1 were expressed in Escherichia coli as GST-fusion proteins and immunized in BALB/c mice. The specificity, subtyping, and affinity of raised antibodies were evaluated by enzyme-linked immunosorbent assays. Cellular immune responses were analyzed by lymphoproliferation assays and cytokine levels produced by splenocytes were detected by cytometry. RESULTS: We found that N-terminal, central regions, and PmMSP119 are strongly immunogenic in mice. After three doses, the induced immune responses remained high for 70 days. While antibodies induced after immunization with N-terminal and central regions showed similar affinities to the target antigens, affinities of IgG against PmMSP119 were higher. All proteins induced similar antibody subclass patterns (predominantly IgG1, IgG2a, and IgG2b), characterizing a mixed Th1/Th2 response. Further, autologous stimulation of splenocytes from immunized mice led to the secretion of IL2 and IL4, independently of the antigen used. Importantly, IgG from P. malariae-exposed individuals reacted against PmMSP1 recombinant proteins with a high specificity. On the other hand, sera from P. vivax or P. falciparum-infected individuals did not react at all against recombinant PmMSP1 proteins. CONCLUSION: Recombinant PmMSP1 proteins are very useful diagnostic markers of P. malariae in epidemiological studies or in the differential diagnosis of malaria caused by this species. Immunization with recombinant PmMSP1 proteins resulted in a significant humoral immune response, which may turn them potential component candidates for a vaccine against P. malariae.

Anopheles (Kerteszia) cruzii (DIPTERA: CULICIDAE) IN PERIDOMICILIARY AREA DURING ASYMPTOMATIC MALARIA TRANSMISSION IN THE ATLANTIC FOREST: MOLECULAR IDENTIFICATION OF BLOOD-MEAL SOURCES INDICATES HUMANS AS PRIMARY INTERMEDIATE HOSTS / Anopheles (Kerteszia) cruzii (Diptera: Culicidae) em área peridomiciliar durante transmissão de malária assintomática na Mata Atlântica: identificação molecular das fontes de repasto sanguíneo indica humanos como hospedeiros intermediários primários

Anopheles (Kerteszia) cruzii has been implicated as the primary vector of human and simian malarias out of the Brazilian Amazon and specifically in the Atlantic Forest regions. The presence of asymptomatic human cases, parasite-positive wild monkeys and the similarity between the parasites infecting them support the discussion whether these infections can be considered as a zoonosis. Although many aspects of the biology of An. cruzii have already been addressed, studies conducted during outbreaks of malaria transmission, aiming at the analysis of blood feeding and infectivity, are missing in the Atlantic Forest. This study was conducted in the location of Palestina, Juquitiba, where annually the majority of autochthonous human cases are notified in the Atlantic Forest of the state of São Paulo. Peridomiciliary sites were selected for collection of mosquitoes in a perimeter of up to 100 m around the residences of human malaria cases. The mosquitoes were analyzed with the purpose of molecular identification of blood-meal sources and to examine the prevalence of Plasmodium. A total of 13,441 females of An. (Ker.) cruzii were collected. The minimum infection rate was calculated at 0.03% and 0.01%, respectively, for P. vivax and P. malariae and only human blood was detected in the blood-fed mosquitoes analyzed. This data reinforce the hypothesis that asymptomatic human carriers are the main source of anopheline infection in the peridomiciliary area, making the probability of zoonotic transmission less likely to happen.

Anopheles (Kerteszia) cruzii é o vetor primário das malárias humana e simiana fora da Amazônia Brasileira e especificamente nas regiões de Mata Atlântica. A presença de casos humanos assintomáticos, macacos silvestres positivos para Plasmodium e a similaridade entre os parasitas que os infectam suportam a discussão se essas infecções podem ser consideradas como zoonoses. Embora muitos aspectos da biologia de An. cruzii já tenham sido abordados, estudos conduzidos durante surtos de transmissão de malária, visando a análise de repasto sanguíneo e infectividade, são ausentes na Mata Atlântica. Este estudo foi conduzido na localidade de Palestina, Juquitiba, Mata Atlântica do Estado de São Paulo, onde anualmente a maioria dos casos humanos autóctones é notificada. Locais em peridomicílio foram selecionados para coleta de mosquitos em um perímetro de até 100 m em torno das residências de casos humanos de malária e da floresta circundante. Os mosquitos foram analisados com o objetivo de identificação molecular das fontes de repasto sanguíneo e para examinar a prevalência de Plasmodium. Um total de 13.441 fêmeas de An. (Ker.) cruzii foi coletado. A taxa de infecção mínima foi calculada a 0,03% e 0,01%, respectivamente, para P. vivax e P. malariae e somente sangue humano foi detectado nos mosquitos analisados que se alimentaram com sangue. Nossos dados reforçam a hipótese de que os portadores humanos assintomáticos são a principal fonte de infecção para os anofelinos na área do peridomicílio, tornando a transmissão zoonótica improvável.

Clinical and molecular aspects of severe malaria

O ciclo eritrocítico do Plasmodium falciparum apresenta uma particularidade em relação às outras espécies de Plasmodium que infectam o homem. Trofozoítas maduros e esquizontes são seqüestrados da circulação periférica devido à adesão de eritrócitos infectados às células endoteliais. Modificações na superfície dos eritrócitos infectados, denominadas "knobs", permitem adesão ao endotélio e a outros eritrócitos. A adesão fornece uma melhor maturação na atmosfera venosa microaerofílica e permite que o parasita escape do clareamento pelo baço, que reconhece a perda de deformabilidade do eritrócito infectado. A adesão ao endotélio ou citoaderência, tem importante função na patogenicidade da doença, causando obstrução de pequenos vasos e contribuindo para danos em muitos órgãos. Citoaderência designa também a adesão de eritrócitos infectados a eritrócitos não infectados, fenômeno amplamente conhecido como "rosetting". Aspectos clínicos da malária grave bem como receptores do hospedeiro e ligantes do parasita envolvidos em citoaderência e "rosetting", são revisados aqui. A proteína de membrana do eritrócito 1 de P. falciparum (PfEMP1) parece ser o principal ligante adesivo dos eritrócitos infectados e será discutida em maiores detalhes. Uma melhor compreensão da função dos receptores do hospedeiro e dos ligantes do parasita no desenvolvimento de diferentes síndromes clínicas é urgentemente necessária para identificar alvos para vacinação visando diminuir as taxas de mortalidade desta doença.

PCR - based diagnosis to evaluate the performance of malaria reference centers

Embora a gota espessa corada por Giemsa (GTS) permaneça o padrão ouro para o diagnóstico de malária, métodos moleculares são mais sensíveis e específicos para detectar parasitas e podem ser utilizados em centros de referência para avaliar o desempenho da microscopia. A descrição das seqüências dos genes ssrRNA de Plasmodium falciparum, P. vivax, P. malariae e P. ovale permitiu o desenvolvimento de uma reação em cadeia da polimerase (PCR) que tem sido utilizada para diferenciar as quatro espécies. O objetivo deste estudo foi determinar as espécies de Plasmodium através de PCR em 190 lâminas positivas de pacientes para verificar a qualidade do diagnóstico realizado no Laboratório de Malária da SUCEN. Considerando somente os 131 resultados positivos em ambas as técnicas, GTS detectou 4,6 de infecçäes mistas e 3,1 de P. malariae enquanto o PCR identificou 19,1 e 13,8, respectivamente.

In vitro evaluation of verapamil and other modulating agents in Brazilian chloroquine-resistant Plasmodium falciparum isolates.

Verapamil, was assayed to record its modulating effect upon Brazilian Plasmodium falciparum isolates resistant to chloroquine. Other cardiovascular drugs known to be modulating agents in resistant malaria and/or multidrug-resistant neoplasias, including nifedipine, nitrendipine, diltiazem and propranolol, were also evaluated. Concentrations similar to those for cardiovascular therapy were used in the in vitro microtechnique for antimalarial drug susceptibility. Intrinsic antiplasmodial activity was observed from the lowest concentrations without a significant modulating action. Other reported modulating agents, such as the antipsychotic drug trifluoperazine and the antidepressants desipramine and imipramine, demonstrated similar responses under the same experimental conditions. Results suggest a much higher susceptibility of Brazilian strains, as well as an indifferent behaviour in relation to modulating agents.

Analysis of genetic relatedness between populations of Aedes aegypti from different geographic regions of São Paulo state, Brazil

RAPD markers have been used for the analysis of genetic differentiation of Aedes aegypti, because they allow the study of genetic relationships among populations. The aim of this study was to identify populations in different geographic regions of the São Paulo State in order to understand the infestation pattern of A. aegypti. The dendrogram constructed with the combined data set of the RAPD patterns showed that the mosquitoes were segregated into two major clusters. Mosquitoes from the Western region of the São Paulo State constituted one cluster and the other was composed of mosquitoes from a laboratory strain and from a coastal city, where the largest Latin American port is located. These data are in agreement with the report on the infestation in the São Paulo State. The genetic proximity was greater between mosquitoes whose geographic origin was closer. However, mosquitoes from the coastal city were genetically closer to laboratory-reared mosquitoes than to field-collected mosquitoes from the São Paulo State. The origin of the infestation in this place remains unclear, but certainly it is related to mosquitoes of origins different from those that infested the West and North region of the State in the 80's

In vitro evaluation of verapamil and other modulating agents in Brazilian chloroquine-resistant Plasmodium falciparum isolates

Verapamil, was assayed to record its modulating effect upon Brazilian Plasmodium falciparum isolates resistant to chloroquine. Other cardiovascular drugs known to be modulating agents in resistant malaria and/or multidrug-resistant neoplasias, including nifedipine, nitrendipine, diltiazem and propranolol, were also evaluated. Concentrations similar to those for cardiovascular therapy were used in the in vitro microtechnique for antimalarial drug susceptibility. Intrinsic antiplasmodial activity was observed from the lowest concentrations without a significant modulating action. Other reported modulating agents, such as the antipsychotic drug trifluoperazine and the antidepressants desipramine and imipramine, demonstrated similar responses under the same experimental conditions. Results suggest a much higher susceptibility of Brazilian strains, as well as an indifferent behaviour in relation to modulating agents

In vitro chloroquine resistance modulation study on fresh isolates of Brazilian Plasmodium falciparum: intrinsic antimalarial activity of phenothiazine drugs

Phenothiazine drugs - fluphenazine, chlorpromazine, methotrimeprazine and trifluoperazine - were evaluated as modulating agents against Brazilian chloroquine-resistant fresh isolates of Plasmodium falciparum. Aiming to simulate therapeutic schedules, chloroquine was employed at the concentration used for sensitive falciparum malaria treatment and anti-psychotic therapeutic concentrations of the phenothiazine drugs were adopted in two-fold serial dilutions. The in vitro microtechnique for drug susceptibility was employed. Unlike earlier reported data, the phenothiazine modulating effect was not observed. However, all the drugs demonstrated intrinsic antiplasmodial activity in concentrations lower than those described in the literature. In addition, IC50 estimates have been shown to be inferior to the usual anti-psychotic therapeutic concentrations. Statistical analysis also suggested an increase in the parasitaemia rate or, even, a predominant antiparasitic effect of phenothiazine over chloroquine when used in combination

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

Association of severe noncerebral Plasmodium falciparum malaria in Brazil with expressed PfEMP1 DBL1 alpha sequences lacking cysteine residues.

BACKGROUND: Cytoadherence and rosetting contribute to the development of severe Plasmodium falciparum malaria. In Brazil,severe falciparum malaria is mostly associated with renal or pulmonary complications and very rarely with cerebral malaria. The most N-terminal DBL1 alpha domain of PfEMP1, a protein encoded by the var multigene family mediates rosetting. We analyzed parasites of Brazilian patients with severe malaria to determine whether there were particular DBL1 alpha var sequences predominantly expressed in such patients. MATERIALS AND METHODS: DBL1 alpha var sequences were obtained from parasites of Brazilian patients with severe and mild malaria and were analyzed by standard bioinformatic programs. Three hundred twenty var DBL1 alpha sequences obtained from 80 Brazilian patients with mild malaria were spotted in high-density filters and hybridized to probes representing predominantly expressed sequences in parasites from patients with severe malaria. A DBL1 alpha domain was expressed in bacteria and used to demonstrate its binding capacity to erythrocytes by immunofluorescence. RESULTS: Forty-three different and unreported DBL1 alpha amino acid sequences were obtained. Sequences predominantly expressed in patients with severe malaria could be subgrouped due to deletions of 1-2-cysteine residues. These sequences were commonly found in the var gene repertoire of parasites from patients with mild malaria, yet they were rarely expressed in these patients. A recombinant protein representing the most abundantly expressed sequence detected in one patient with severe malaria bound directly to uninfected erythrocytes. CONCLUSIONS: This is the first report showing an association of severe noncerebral malaria from Brazil with particular DBL1 alpha sequences.

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

In vitro evaluation of erythromycin in chloroquine resistant brazilian P. falciparum freshly isolates: modulating effect and antimalarial activity evidence

Erythromycin, a reversal agent in multidrug-resistant cancer, was assayed in chloroquine resistance modulation. The in vitro microtechnique for drug susceptibility was employed using two freshly isolates of Plasmodium falciparum from North of Brazil. The antimalarial effect of the drug was confirmed, with an IC50 estimates near the usual antimicrobial therapy concentration, and a significant statistical modulating action was observed for one isolate