Asymptomatic carriers of Plasmodium spp. as infection source for malaria vector mosquitoes in the Brazilian Amazon

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. © 2005 Entomological Society of America.

Imported Malaria In A Non-endemic Area: The Experience Of The University Of Campinas Hospital In The Brazilian Southeast.

Although malaria in Brazil almost exclusively occurs within the boundaries of the Amazon Region, some concerns are raised regarding imported malaria to non-endemic areas of the country, notably increased incidence of complications due to delayed diagnoses. However, although imported malaria in Brazil represents a major health problem, only a few studies have addressed this subject. A retrospective case series is presented in which 263 medical charts were analysed to investigate the clinical and epidemiological characterization of malaria cases that were diagnosed and treated at Hospital & Clinics, State University of Campinas between 1998 and 2011. Amongst all medical charts analysed, 224 patients had a parasitological confirmed diagnosis of malaria. Plasmodium vivax and Plasmodium falciparum were responsible for 67% and 30% of the infections, respectively. The majority of patients were male (83%) of a productive age (median, 37 years old). Importantly, severe complications did not differ significantly between P. vivax (14 cases, 9%) and P. falciparum (7 cases, 10%) infections. Severe malaria cases were frequent among imported cases in Brazil outside of the Amazon area. The findings reinforce the idea that P. vivax infections in Brazil are not benign, regardless the endemicity of the area studied. Moreover, as the hospital is located in a privileged site, it could be used for future studies of malaria relapses and primaquine resistance mechanisms. Finally, based on the volume of cases treated and the secondary complications, referral malaria services are needed in the non-endemic areas of Brazil for a rapid and efficient and treatment.

Epidemiological and ecological aspects related to malaria in the area of influence of the lake at Porto Primavera dam, in western São Paulo State, Brazil

A study was carried out in the area of influence of the Porto Primavera Hydroelectric Power Station, in western São Paulo State, to investigate ecological and epidemiological aspects of malaria in the area and monitor the profile of the anopheline populations following the environmental changes brought about by the construction of the lake. Mosquitoes captured were analyzed by standardized indicator species analysis (ISA) before and during different flooding phases (253 m and 257 m elevations). The local human population was studied by means of parasitological (thin/thick blood smears), molecular (PCR) and serological tests. Serological tests consisted of Enzyme Linked Immunosorbent Assay (ELISA) with synthetic peptides of the circumsporozoite protein (CSP) from classic Plasmodium vivax, P. vivax variants (VK247 and "vivax-like"), P. malariae and P. falciparum and Indirect Immunofluorescence Assay (IFA) with asexual forms of P. vivax, P. malariae and P. falciparum. The results of the entomological survey indicated that, although the Anopheles darlingi population increased after the flooding, the population density remained very low. No malaria, parasite infection or DNA was detected in the inhabitants of the study area. However, there was a low frequency of antibodies against asexual forms and a significant prevalence of antibodies against P. vivax, P. vivax variants, P. falciparum and P. malariae; the presence of these antibodies may result from recent or less recent contact with human or simian Plasmodium (a parallel study in the same area revealed the existence of a sylvatic cycle). Nevertheless, these results suggest that, as in other places where malaria is present and potential vectors circulate, the local epidemiological conditions observed could potentially support the transmission of malaria in Porto Primavera Lake if infected individuals are introduced in sufficient numbers. Further studies are required to elucidate the phenomena described in this paper.

Entomological characterization and natural infection of anophelines in an area of the Atlantic Forest with autochthonous malaria cases in mountainous region of Espírito Santo State, Brazil

No Espírito Santo, os casos de malária autóctone estão distribuídos na região serrana próximo aos fragmentos de Mata Atlântica. Uma vez que alguns aspectos da doença são obscuros, a detecção das possíveis espécies de vetores pode auxiliar na elucidação de incertezas epidemiológicas. Estudos entomológicos e de infecção natural foram realizados com anofelinos (Diptera: Culicidae) capturados no município de Santa Tereza, ES. Capturas mensais foram realizadas de março de 2004 a fevereiro de 2006. Armadilhas CDC-CO2 foram utilizadas do crepúsculo (18:00h) ao amanhecer (6:00h), para capturar anofelinos nos seguintes habitats: próximo ao domicílio e área aberta (solo), margem e interior da mata (solo e copa). Armadilhas Shannon também foram utilizadas nos mesmos locais que as de CDC-CO2. Capturou-se o total de 2.290 anofelinos distribuídos em 10 espécies. A maior frequência relativa foi de Anopheles (Kerteszia) cruzii Dyar & Knab / A.(K.) homunculus Komp, sendo a maioria capturada em CDC-CO2 instalada na copa da mata. A principal espécie capturada em armadilha Shannon foi A.(Nyssorhynchus) strodei Root. O maior número de anofelinos foi capturado entre julho e setembro das 18:00h às 22:00h. Provavelmente A.(K.) cruzii é responsável pela transmissão da malária dentro ou próximo aos fragmentos de Mata Atlântica. Entretanto, a participação de outras espécies não pode ser ignorada, visto que 53 por cento da amostragem foi constituída pelo subgênero Nyssorhynchus. A detecção de Plasmodium vivax no tórax de A. cruzii, A. parvus (Chagas) e A. galvaoi Causey, Deane & Deane por meio de PCR reforça esse argumento

Spatial patterns of malaria in a land reform colonization project, Juruena municipality, Mato Grosso, Brazil

Background: In Brazil, 99% of malaria cases are concentrated in the Amazon, and malaria's spatial distribution is commonly associated with socio-environmental conditions on a fine landscape scale. In this study, the spatial patterns of malaria and its determinants in a rural settlement of the Brazilian agricultural reform programme called ""Vale do Amanhecer"" in the northern Mato Grosso state were analysed. Methods: In a fine-scaled, exploratory ecological study, geocoded notification forms corresponding to malaria cases from 2005 were compared with spectral indices, such as the Normalized Difference Vegetation Index (NDVI) and the third component of the Tasseled Cap Transformation (TC_3) and thematic layers, derived from the visual interpretation of multispectral TM-Landsat 5 imagery and the application of GIS distance operators. Results: Of a total of 336 malaria cases, 102 (30.36%) were caused by Plasmodium falciparum and 174 (51.79%) by Plasmodium vivax. Of all the cases, 37.6% (133 cases) were from residents of a unique road. In total, 276 cases were reported for the southern part of the settlement, where the population density is higher, with notification rates higher than 10 cases per household. The local landscape mostly consists of open areas (38.79 km(2)). Training forest occupied 27.34 km(2) and midsize vegetation 7.01 km(2). Most domiciles with more than five notified malaria cases were located near areas with high NDVI values. Most domiciles (41.78%) and malaria cases (44.94%) were concentrated in areas with intermediate values of the TC_3, a spectral index representing surface and vegetation humidity. Conclusions: Environmental factors and their alteration are associated with the occurrence and spatial distribution of malaria cases in rural settlements.

Genetic polymorphisms in TLR4, CR1 and Duffy genes are not associated with malaria resistance in patients from Baixo Amazonas region, Brazil

The main purpose of this research was to analyze the relation of the genetic polymorphisms frequently expressed by antigen-presenting cells, erythrocytes and malaria susceptibility/resistance with the human malaria infection cases. The sample used consisted of 23 Plasmodium vivax ( Pv)- and P. falciparum ( Pf)-infected patients, and 21 healthy individuals as a control group, from the Baixo Amazonas population in Para, Brazil. The Asp299Gly polymorphisms in the Toll-like receptor 4 ( TLR4), and Gly42Asp, Arg89Cys, Ala100Thr, and T-33C in the Duffy gene ( FY) were analyzed by restriction fragment length polymorphism-polymerase chain reaction. The Lys1590Glu and Arg1601Gly polymorphisms in the complement receptor type 1 (CR1) were analyzed by DNA sequencing. According to the results obtained and statistical analysis considering a significance level or alpha = 0.01, we conclude that the low heterozygote frequency (2.27%) for the Asp299Gly mutation, detected in the TLR4 gene, is not related to the Pv and Pf infections in the patients analyzed. Also, the promoter region GATA-1 analysis of the FY gene in the Pv-infected patients showed that the heterozygote frequency for the T-33C mutation (11.36% of the infected patients and 20.45% of the control patients) is not related to infection resistance. Regarding the CR1 gene, the observed heterozygote frequency (9.09%) for the Arg1601Gly mutation in Pf-infected patients when compared to heterozygote frequency in the control group (18.18%) suggests that there is no correlation with infection resistance.

Cost effectiveness of OptiMal (R) rapid diagnostic test for malaria in remote areas of the Amazon Region, Brazil

Background: In areas with limited structure in place for microscopy diagnosis, rapid diagnostic tests (RDT) have been demonstrated to be effective. Method: The cost-effectiveness of the Optimal (R) and thick smear microscopy was estimated and compared. Data were collected on remote areas of 12 municipalities in the Brazilian Amazon. Data sources included the National Malaria Control Programme of the Ministry of Health, the National Healthcare System reimbursement table, hospitalization records, primary data collected from the municipalities, and scientific literature. The perspective was that of the Brazilian public health system, the analytical horizon was from the start of fever until the diagnostic results provided to patient and the temporal reference was that of year 2006. The results were expressed in costs per adequately diagnosed cases in 2006 U. S. dollars. Sensitivity analysis was performed considering key model parameters. Results: In the case base scenario, considering 92% and 95% sensitivity for thick smear microscopy to Plasmodium falciparum and Plasmodium vivax, respectively, and 100% specificity for both species, thick smear microscopy is more costly and more effective, with an incremental cost estimated at US$ 549.9 per adequately diagnosed case. In sensitivity analysis, when sensitivity and specificity of microscopy for P. vivax were 0.90 and 0.98, respectively, and when its sensitivity for P. falciparum was 0.83, the RDT was more cost-effective than microscopy. Conclusion: Microscopy is more cost-effective than OptiMal (R) in these remote areas if high accuracy of microscopy is maintained in the field. Decision regarding use of rapid tests for diagnosis of malaria in these areas depends on current microscopy accuracy in the field.

Hepatitis B Infection Is Associated with Asymptomatic Malaria in the Brazilian Amazon

Background: Areas that are endemic for malaria are also highly endemic for hepatitis B virus (HBV) infection. Nevertheless, it is unknown whether HBV infection modifies the clinical presentation of malaria. This study aimed to address this question. Methodology and Findings: An observational study of 636 individuals was performed in Rondonia, western Amazon, Brazil between 2006 and 2007. Active and passive case detections identified Plasmodium infection by field microscopy and nested Polymerase Chain Reaction (PCR). HBV infections were identified by serology and confirmed by real-time PCR. Epidemiological information and plasma cytokine profiles were studied. The data were analyzed using adjusted multinomial logistic regression. Plasmodium-infected individuals with active HBV infection were more likely to be asymptomatic (OR: 120.13, P < 0.0001), present with lower levels of parasitemia and demonstrate a decreased inflammatory cytokine profile. Nevertheless, co-infected individuals presented higher HBV viremia. Plasmodium parasitemia inversely correlated with plasma HBV DNA levels (r=-0.6; P=0.0003). Conclusion: HBV infection diminishes the intensity of malaria infection in individuals from this endemic area. This effect seems related to cytokine balance and control of inflammatory responses. These findings add important insights to the understanding of the factors affecting the clinical outcomes of malaria in endemic regions.

An algorithm to predict 3 ' intron splice sites in Plasmodium falciparum genomic sequences

A new algorithm, PfAGSS, for predicting 3' splice sites in Plasmodium falciparum genomic sequences is described. Application of this program to the published P. falciparum chromosome 2 and 3 data suggests that existing programs result in a high error rate in assigning 3' intron boundaries. (C) 2001 Elsevier Science B.V. All rights reserved.

Extensive multiple test centre evaluation of the VecTest malaria antigen panel assay

To determine which species and populations of Anopheles transmit malaria in any given situation, immunological assays for malaria sporozoite antigen can replace traditional microscopical examination of freshly dissected Anopheles. We developed a wicking assay for use with mosquitoes that identifies the presence or absence of specific peptide epitopes of circumsporozoite (CS) protein of Plasmodium falciparum and two strains of Plasmodium vivax (variants 210 and 247). The resulting assay (VecTest(TM) Malaria) is a rapid, one-step procedure using a 'dipstick' test strip capable of detecting and distinguishing between P. falciparum and P. vivax infections in mosquitoes. The objective of the present study was to test the efficacy, sensitivity, stability and field-user acceptability of this wicking dipstick assay. In collaboration with 16 test centres world-wide, we evaluated more than 40 000 units of this assay, comparing it to the standard CS ELISA. The 'VecTest(TM) Malaria' was found to show 92% sensitivity and 98.1% specificity, with 97.8% accuracy overall. In accelerated storage tests, the dipsticks remained stable for >15 weeks in dry conditions up to 45degreesC and in humid conditions up to 37degreesC. Evidently, this quick and easy dipstick test performs at an acceptable level of reliability and offers practical advantages for field workers needing to make rapid surveys of malaria vectors.

Malaria at Humaita county, Amazonas state, Brazil: XVII — immune response in patients with Plasmodium falciparum according to gametocytes

In August 1983 the Authors studied 36 patients with Plasmodium falciparum malaria and 14 normal individuals born in Humaita region who had never had malaria, had no spleen enlargement and had negative parasitemia as well as passive hemagglutination. Medical histories were obtained and complete physical examination were performed in all of them just as blood tests, parasite density and lymphocyte typing. The lymphocytes were separated and then frozen in liquid nitrogen for later typing by rosette formation. The patients were divided in two groups according to the presence (13 patients) or abscence (23 patients) of gametocytes before treatment. Severe malaria was predominant in the group without gametocytes. The results showed a decrease in the T-cell numbers in Plasmodium falciparum acute malaria patients both with or without gametocytes before the treatment, while B-cell numbers were normal only in the patients with gametocytes. These observations as like as those previously reported by the Authors, permit to associate the presence of gametocytes in peripheral blood and normal number of B-cells in patients with mild Plasmodium falciparum malaria.

Retrospective study of malaria prevalence and Anopheles genus in the area of influence of the binational Itaipu reservoir

The importance of hydroelectric dams beside the human interchange in the maintenance of malarious foci and the occurrence of the Anopheles genus on the Binational Itaipu Reservoir were the main points of this retrospective study. Data were collected from existing registrations at National, State and Municipal Health Departments and literature systematic overview, from January 1984 to December 2003. The occurrence of some outbreak of malaria, mainly by Plasmodium vivax, and the prevalence of species of the Anopheles genus different from Anopheles darlingi in the region are discussed. The malaria in the left bank of Paraná River is a focal problem, which must be approached locally through health, educational and social actions to prevent the continuity of outbreaks in the area. Concomitantly, it is necessary to plan and apply effective surveillance measures in the influence area of the Itaipu Reservoir.

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.

Epidemiological and ecological aspects related to malaria in the area of influence of the lake at Porto Primavera dam, in western São Paulo State, Brazil

A study was carried out in the area of influence of the Porto Primavera Hydroelectric Power Station, in western São Paulo State, to investigate ecological and epidemiological aspects of malaria in the area and monitor the profile of the anopheline populations following the environmental changes brought about by the construction of the lake. Mosquitoes captured were analyzed by standardized indicator species analysis (ISA) before and during different flooding phases (253 m and 257 m elevations). The local human population was studied by means of parasitological (thin/thick blood smears), molecular (PCR) and serological tests. Serological tests consisted of Enzyme Linked Immunosorbent Assay (ELISA) with synthetic peptides of the circumsporozoite protein (CSP) from classic Plasmodium vivax, P. vivax variants (VK247 and "vivax-like"), P. malariae and P. falciparum and Indirect Immunofluorescence Assay (IFA) with asexual forms of P. vivax, P. malariae and P. falciparum. The results of the entomological survey indicated that, although the Anopheles darlingi population increased after the flooding, the population density remained very low. No malaria, parasite infection or DNA was detected in the inhabitants of the study area. However, there was a low frequency of antibodies against asexual forms and a significant prevalence of antibodies against P. vivax, P. vivax variants, P. falciparum and P. malariae; the presence of these antibodies may result from recent or less recent contact with human or simian Plasmodium (a parallel study in the same area revealed the existence of a sylvatic cycle). Nevertheless, these results suggest that, as in other places where malaria is present and potential vectors circulate, the local epidemiological conditions observed could potentially support the transmission of malaria in Porto Primavera Lake if infected individuals are introduced in sufficient numbers. Further studies are required to elucidate the phenomena described in this paper.

The monitoring of hematopoietic stem cell transplant donors and recipients from endemic areas for malaria

Malaria is an unusual complication after hematopoietic stem cell transplantation in non-endemic countries. However, transplant candidates, recipients and donors living in endemic regions frequently report previous episodes of malaria. This fact could represent an important risk for immunosuppressed recipients that could develop severe malaria cases. We report a case of hematopoietic stem cell transplant (HSCT) in which the donor had a history of previous malaria, and close monitoring was performed before and after procedure by parasitological and molecular tests. The donor presented Plasmodium vivax in thick blood smears one month after transplant and was treated according to Brazilian Health Ministry guidelines. The polymerase chain reaction (PCR) was able to detect malaria infection in the donor one week earlier than thick blood film. Even without positive results, the recipient was pre-emptively treated with chloroquine in order to prevent the disease. We highlight the importance of monitoring recipients and donors in transplant procedures with the aim of reducing the risk of malaria transmission.