Population genetic structure of the major malaria vector Anopheles darlingi (Diptera: Culicidae) from the Brazilian Amazon, using microsatellite markers

The population genetic structure of Anopheles darlingi, the major human malaria vector in the Neotropics, was examined using seven microsatellite loci from nine localities in central and western Amazonian Brazil. High levels of genetic variability were detected (5-25 alleles per locus; H E = 0.519-0.949). There was deviation from Hardy-Weinberg Equilibrium for 59.79% of the tests due to heterozygote deficits, while the analysis of linkage disequilibrium was significant for only two of 189 (1.05%) tests, most likely caused by null alleles. Genetic differentiation (F ST = 0.001-0.095; Nm = 4.7-363.8) indicates that gene flow is extensive among locations < 152 km apart (with two exceptions) and reduced, but not absent, at a larger geographic scale. Genetic and geographic distances were significantly correlated (R² = 0.893, P < 0.0002), supporting the isolation by distance (IBD) model. The overall estimate of Ne was 202.4 individuals under the linkage disequilibrium model, and 8 under the heterozygote excess model. Analysis of molecular variance showed that nearly all variation (~ 94%) was within sample locations. The UPGMA phenogram clustered the samples geographically, with one branch including 5/6 of the state of Amazonas localities and the other branch the Acre, Rondônia, and remaining Amazonas localities. Taken together, these data suggest little genetic structure for An. darlingi from central and western Amazonian Brazil. These findings also imply that the IBD model explains nearly all of the differentiation detected. In practical terms, populations of An. darlingi at distances < 152 km should respond similarly to vector control measures, because of high gene flow.

Clinical spectrum of uncomplicated malaria in semi-immune Amazonians: beyond the " symptomatic " vs " asymptomatic " dichotomy

We analyzed prospectively 326 laboratory-confirmed, uncomplicated malarial infections (46.3% due to Plasmodium vivax, 35.3% due to P. falciparum, and 18.4% mixed-species infections) diagnosed in 162 rural Amazonians aged 5-73 years. Thirteen symptoms (fever, chills, sweating, headache, myalgia, arthralgia, abdominal pain, nausea, vomiting, dizziness, cough, dyspnea, and diarrhea) were scored using a structured questionnaire. Headache (59.8%), fever (57.1%), and myalgia (48.4%) were the most frequent symptoms. Ninety-six (29.4%) episodes, all of them diagnosed during cross-sectional surveys of the whole study population (96.9% by molecular technique only), were asymptomatic. Of 93 symptom-less infections left untreated, only 10 became symptomatic over the next two months following diagnosis. Fever was perceived as " intense " in 52.6% of 230 symptomatic malaria episodes, with no fever reported in 19.1% episodes although other symptoms were present. We found significant differences in the prevalence and perceived intensity of fever and other clinical symptoms in relation to parasite load at the time of diagnosis and patient's age, cumulative exposure to malaria, recent malaria morbidity, and species of malaria parasite. These factors are all likely to affect the effectiveness of malaria control strategies based on active or passive detection of febrile subjects in semi-immune populations.

An ecoregional classification for the state of Roraima, Brazil: the importance of landscape in malaria biology

Understanding the different background landscapes in which malaria transmission occurs is fundamental to understanding malaria epidemiology and to designing effective local malaria control programs. Geology, geomorphology, vegetation, climate, land use, and anopheline distribution were used as a basis for an ecological classification of the state of Roraima, Brazil, in the northern Amazon Basin, focused on the natural history of malaria and transmission. We used unsupervised maximum likelihood classification, principal components analysis, and weighted overlay with equal contribution analyses to fine-scale thematic maps that resulted in clustered regions. We used ecological niche modeling techniques to develop a fine-scale picture of malaria vector distributions in the state. Eight ecoregions were identified and malaria-related aspects are discussed based on this classification, including 5 types of dense tropical rain forest and 3 types of savannah. Ecoregions formed by dense tropical rain forest were named as montane (ecoregion I), submontane (II), plateau (III), lowland (IV), and alluvial (V). Ecoregions formed by savannah were divided into steppe (VI, campos de Roraima), savannah (VII, cerrado), and wetland (VIII, campinarana). Such ecoregional mappings are important tools in integrated malaria control programs that aim to identify specific characteristics of malaria transmission, classify transmission risk, and define priority areas and appropriate interventions. For some areas, extension of these approaches to still-finer resolutions will provide an improved picture of malaria transmission patterns.

Prevalence and level of antibodies to the circumsporozoite protein of human malaria parasites in five states of the Amazon region of Brazil

The aim of this study was to determine the prevalence of malaria infection and antibodies against the repetitive epitopes of the circumsporozoite (CS) proteins of Plasmodium falciparum, P. malariae, P. vivax VK210, P. vivax VK247, and P. vivax-like in individuals living in the states of Rondônia, Pará, Mato Grosso, Amazonas, and Acre. Active malaria transmission was occurring in all studied sites, except in Acre. P. falciparum was the predominant species in Pará and Rondônia and P. vivax in Mato Grosso. Infection by P. malariae was low but this Plasmodium species was detected in Rondônia (3.5%), Mato Grosso (2.5%), and Pará (0.8%). High prevalence and levels of serological reactivity against the CS repeat peptides of P. falciparum were detected in Rondônia (93%) and Pará (85%). Sera containing antibodies against the CS repeat of P. malariae occurred more frequently in Rondônia (79%), Pará (76%), and Amazonas (68%). Antibodies against the repeat epitope of the standard CS protein of P. vivax VK210, P. vivax VK247, and P. vivax-like were more frequent in Rondônia, Pará, and Mato Grosso. The high frequency of reactions to P. malariae in most of the areas suggests that the infection by this Plasmodium species has been underestimated in Brazil.

Coadaptation and malaria control

Malaria emerges from a disequilibrium of the system 'human-plasmodium-mosquito' (HPM). If the equilibrium is maintained, malaria does not ensue and the result is asymptomatic plasmodium infection. The relationships among the components of the system involve coadaptive linkages that lead to equilibrium. A vast body of evidence supports this assumption, including the strategies involved in the relationships between plasmodium and human and mosquito immune systems, and the emergence of resistance of plasmodia to antimalarial drugs and of mosquitoes to insecticides. Coadaptive strategies for malaria control are based on the following principles: (1) the system HPM is composed of three highly complex and dynamic components, whose interplay involves coadaptive linkages that tend to maintain the equilibrium of the system; (2) human and mosquito immune systems play a central role in the coadaptive interplay with plasmodium, and hence, in the mainten-ance of the system's equilibrium; the under- or overfunction of human immune system may result in malaria and influence its severity; (3) coadaptation depends on genetic and epigenetic phenomena occurring at the interfaces of the components of the system, and may involve exchange of infectrons (genes or gene fragments) between the partners; (4) plasmodia and mosquitoes have been submitted to selective pressures, leading to adaptation, for an extremely long while and are, therefore, endowed with the capacity to circumvent both natural (immunity) and artificial (drugs, insecticides, vaccines) measures aiming at destroying them; (5) since malaria represents disequilibrium of the system HPM, its control should aim at maintaining or restoring this equilibrium; (6) the disequilibrium of integrated systems involves the disequilibrium of their components, therefore the maintenance or restoration of the system's equilibrium depend on the adoption of integrated and coordinated measures acting on all components, that means, panadaptive strategies. Coadaptive strategies for malaria control should consider that: (1) host immune response has to be induced, since without it, no coadaptation is attained; (2) the immune response has to be sustained and efficient enough to avoid plasmodium overgrowth; (3) the immune response should not destroy all parasites; (4) the immune response has to be well controlled in order to not harm the host. These conditions are mostly influenced by antimalarial drugs, and should also be taken into account for the development of coadaptive malaria vaccines.

Anti-malaria humoral responses in children exposed to Plasmodium falciparum and Schistosoma haematobium

Antibody responses directed against the Plasmodium falciparum antigens, total extract, anti-merozoite surface protein-3 (MSP3b) and glutamate-rich protein (Glurp-R0) were studied in 42 children exposed to both Schistosoma haematobium and P. falciparum infections. The association between levels of the anti-malaria IgG subclasses and IgM with host age, sex, schistosome infection intensity and schistosome specific antibodies was studied before chemotherapeutic treatment of schistosome infections. This showed a significant negative association between schistosome infection intensity and levels of IgG1, IgG3, and IgG4 directed against malaria total extract antigen, and a positive association between levels of anti-schistosome soluble egg antigen IgG2, IgG3, and IgG4 and levels of the same subclasses directed against malaria total extract antigens. The effect of treating schistosome infections with praziquantel on malaria specific responses was also studied. This treatment resulted in increases in significant IgG4 levels against MSP3b and IgM against Glurp R0. Treatment also resulted in a significant decrease in IgG4 levels against Glurp R0. Host age, sex or pre-treatment infection intensity was not associated with the magnitude of change in the two IgG4 responses while males showed a significantly higher increase in levels of IgM. The results suggest cross reactivity between schistosome and malaria antigens in this population.

Merozoite surface protein 2 allelic variation influences the specific antibody response during acute malaria in individuals from a Brazilian endemic area

The antibody response to Plasmodium falciparum parasites of naturally infected population is critical to elucidate the role of polymorphic alleles in malaria. Thus, we evaluated the impact of antigenic diversity of repetitive and family dimorphic domains of the merozoite surface protein 2 (MSP-2) on immune response of 96 individuals living in Peixoto de Azevedo (MT-Brazil), by ELISA using recombinant MSP-2 proteins. The majority of these individuals were carrying FC27-type infections. IgG antibody responses were predominantly directed to FC27 parasites and were correlated to the extension of polymorphism presented by each MSP-2 region. This finding demonstrated the impact of the genetic polymorphism on antibody response and therefore, its importance on malaria vaccine efficacy.

Population structure of the malaria vector Anopheles darlingi in Rondônia, Brazilian Amazon, based on mitochondrial DNA

Anopheles darlingi is the most important Brazilian malaria vector, with a widespread distribution in the Amazon forest. Effective strategies for vector control could be better developed through knowledge of its genetic structure and gene flow among populations, to assess the vector diversity and competence in transmitting Plasmodium. The aim of this study was to assess the genetic diversity of An. darlingi collected at four locations in Porto Velho, by sequencing a fragment of the ND4 mitochondrial gene. From 218 individual mosquitoes, we obtained 20 different haplotypes with a diversity index of 0.756, equivalent to that found in other neotropical anophelines. The analysis did not demonstrate significant population structure. However, haplotype diversity within some populations seems to be over-represented, suggesting the presence of sub-populations, but the presence of highly represented haplotypes complicates this analysis. There was no clear correlation among genetic and geographical distance and there were differences in relation to seasonality, which is important for malarial epidemiology.

Patterns of co-association of C-reactive protein and nitric oxide in malaria in endemic areas of Iran

In addition to numerous immune factors, C-reactive protein (CRP) and nitric oxide (NO) are believed to be molecules of malaria immunopathology. The objective of this study was to detect CRP and NO inductions by agglutination latex test and Griess microassay respectively in both control and malaria groups from endemic areas of Iran, including Southeastern (SE) (Sistan & Balouchestan, Hormozgan, Kerman) and Northwestern (NW) provinces (Ardabil). The results indicated that CRP and NO are produced in all malaria endemic areas of Iran. In addition, more CRP and NO positive cases were observed amongst malaria patients in comparison with those in control group. A variable co-association of CRP/NO production were detected between control and malaria groups, which depended upon the malaria endemic areas and the type of plasmodia infection. The percentage of CRP/NO positive cases was observed to be lower in NW compare to SE region, which may be due to the different type of plasmodium in the NW (Plasmodium vivax) with SE area (P. vivax, Plasmodium falciparum, mixed infection). The fluctuations in CRP/NO induction may be consistent with genetic background of patients. Although, CRP/NO may play important role in malaria, their actual function and interaction in clinical forms of disease remains unclear.

Evaluation of a malaria antibody enzyme immunoassay for use in blood screening

Transfusion-transmitted malaria is rare, but it may produce severe problem in the safety of blood transfusion due to the lack of reliable procedure to evaluate donors potentially exposed to malaria. Here, we evaluated a new enzyme-linked immunosorbent assay malaria antibody test (ELISA malaria antibody test, DiaMed, Switzerland) to detect antibodies to Plasmodium vivax (the indigenous malaria) in the blood samples in the Republic of Korea (ROK). Blood samples of four groups were obtained and analyzed; 100 samples from P.vivax infected patients, 35 from recovery patients, 366 from normal healthy individuals, and 325 from domestic travelers of non-endemic areas residents to risky areas of ROK. P.vivax antibody levels by ELISA were then compared to the results from microscopic examination and polymerase chain reaction (PCR) test. As a result, the ELISA malaria antibody test had a clinical sensitivity of 53.0% and a clinical specificity of 94.0% for P.vivax. Twenty out of 325 domestic travelers (6.2%) were reactive and 28 cases (8.6%) were doubtful. Of the reactive and doubtful cases, only two were confirmed as acute malaria by both microscopy and PCR test. Thus we found that the ELISA malaria antibody test was insufficiently sensitive for blood screening of P.vivax in ROK.

Chloroquine resistant Plasmodium falciparum malaria in Osogbo Nigeria: efficacy of amodiaquine + sulfadoxine-pyrimethamine and chloroquine + chlorpheniramine for treatment

Chloroquine (CQ) resistance in Plasmodium falciparum contributes to increasing malaria-attributable morbidity and mortality in Sub-Saharan Africa. Despite a change in drug policy, continued prescription of CQ did not abate. Therefore the therapeutic efficacy of CQ in uncomplicated falciparum malaria patients was assessed in a standard 28-day protocol in 116 children aged between six and 120 months in Osogbo, Southwest Nigeria. Parasitological and clinical assessments of response to treatment showed that 72 (62.1%) of the patients were cured and 44 (37.9%) failed the CQ treatment. High initial parasite density and young age were independent predictors for early treatment failure. Out of the 44 patients that failed CQ, 24 received amodiaquine + sulphadoxine/pyrimethamine (AQ+SP) and 20 received chlorpheniramine + chloroquine (CH+CQ) combinations. Mean fever clearance time in those treated with AQ+SP was not significantly different from those treated with CH+CQ (p = 0.05). There was no significant difference in the mean parasite density of the two groups. The cure rate for AQ+SP group was 92% while those of CH+CQ was 85%. There was a significant difference in parasite clearance time (p = 0.01) between the two groups. The 38% treatment failure for CQ reported in this study is higher than the 10% recommended by World Health Organization in other to effect change in antimalarial treatment policy. Hence we conclude that CQ can no more be solely relied upon for the treatment of falciparum malaria in Osogbo, Nigeria. AQ+SP and CH+CQ are effective in the treatment of acute uncomplicated malaria and may be considered as useful alternative drugs in the absence of artemisinin-based combination therapies.

Insecticide treated mosquito nets for malaria control in India-experience from a tribal area on operational feasibility and uptake

The study assessed the operational feasibility and acceptability of insecticide-treated mosquito nets (ITNs) in one Primary Health Centre (PHC) in a falciparum malaria endemic district in the state of Orissa, India, where 74% of the people are tribes and DDT indoor residual spraying had been withdrawn and ITNs introduced by the National Vector Borne Disease Control Programme. To a population of 63,920, 24,442 ITNs were distributed free of charge through 101 treatment centers during July-August 2002. Interview of 1,130, 1,012 and 126 respondents showed that the net use rates were 80%, 74% and 55% in the cold, rainy and summer seasons, respectively. Since using ITNs, 74.5-76.6% of the respondents observed reduction of mosquito bites and 7.2-32.1% reduction of malaria incidence; 37% expressed willingness to buy ITNs if the cost was lower and they were affordable. Up to ten months post-treatment, almost 100% mortality of vector mosquitoes was recorded on unwashed and washed nets (once or twice). Health workers re-treated the nets at the treatment centers eight months after distribution on a cost-recovery basis. The coverage reported by the PHC was only 4.2%, mainly because of unwillingness of the people to pay for re-treatment and to go to the treatment centers from their villages. When the re-treatment was continued at the villages involving personnel from several departments, the coverage improved to about 90%.Interview of 126 respondents showed that among those who got their nets re-treated, 81.4% paid cash for the re-treatment and the remainder were reluctant to pay. Majority of those who paid said that they did so due to the fear that if they did not do so they would lose benefits from other government welfare schemes. The 2nd re-treatment was therefore carried out free of charge nine months after the 1st re-treatment and thus achieved coverage of 70.4%. The study showed community acceptance to use ITNs as they perceived the benefit. Distribution and re-treatment of nets was thus possible through the PHC system, if done free of charge and when personnel from different departments, especially those at village level, were involved.

The role of platelet and plasma markers of antioxidant status and oxidative stress in thrombocytopenia among patients with vivax malaria

Malaria remains an important health problem in tropical countries like Brazil. Thrombocytopenia is the most common hematological disturbance seen in malarial infection. Oxidative stress (OS) has been implicated as a possible mediator of thrombocytopenia in patients with malaria. This study aimed to investigate the role of OS in the thrombocytopenia of Plasmodium vivax malaria through the measurement of oxidant and antioxidant biochemical markers in plasma and in isolated platelets. Eighty-six patients with P. vivax malaria were enrolled. Blood samples were analyzed for total antioxidant and oxidant status, albumin, total protein, uric acid, zinc, magnesium, bilirubin, total thiols, glutathione peroxidase (GPx), malondialdehyde (MDA), antibodies against mildly oxidized low-density lipoproteins (LDL-/nLDL ratio) and nitrite/nitrate levels in blood plasma and GPx and MDA in isolated platelets. Plasma MDA levels were higher in thrombocytopenic (TCP) (median 3.47; range 1.55-12.90 µmol/L) compared with the non-thrombocytopenic (NTCP) patients (median 2.57; range 1.95-8.60 µmol/L). Moreover, the LDL-/nLDL autoantibody ratio was lower in TCP (median 3.0; range 1.5-14.8) than in NTCP patients (median 4.0; range 1.9-35.5). Finally, GPx and MDA were higher in the platelets of TPC patients. These results suggest that oxidative damage of platelets might be important in the pathogenesis of thrombocytopenia found in P. vivax malaria as indicated by alterations of GPx and MDA.

Improving the production of the denatured recombinant N-terminal domain of rhoptry-associated protein 2 from a Plasmodium falciparum target in the pathology of anemia in falciparum malaria

Rhoptry-associated protein 2 (RAP2) is known to be discharged from rhoptry onto the membrane surface of infected and uninfected erythrocytes (UEs) ex vivo and in vitro and this information provides new insights into the understanding of the pathology of severe anemia in falciparum malaria. In this study, a hexahistidine-tagged recombinant protein corresponding to residues 5-190 of the N-terminal of Plasmodium falciparum RAP2 (rN-RAP2) was produced using a new method of solubilization and purification. Expression was induced with D-lactose, a less expensive alternative inducer to the more common isopropyl-²-D-thio-galactopyranosidase. The recombinant protein was purified using two types of commercially-available affinity columns, iminodiacetic and nitrilotriacetic. rN-RAP2 had immunogenic potential, since it induced high titers of anti-RAP2 antibodies in mice. These antibodies recognized full-length RAP2 prepared from Triton X-100 extracts from two strains of P. falciparum. In fact, the antibody recognized a 29-kDa product of RAP2 cleavage as well as 82 and 70-kDa products of RAP1 cleavage. These results indicate that the two antigens share sequence epitopes. Our expressed protein fragment was shown to contain a functional epitope that is also present in rhoptry-derived ring surface protein 2 which attaches to the surface of both infected and UEs and erythroid precursor cells in the bone marrow of malaria patients. Serum from malaria patients who developed anemia during infection recognized rN-RAP2, suggesting that this protein fragment may be important for epidemiological studies investigating whether immune responses to RAP2 exacerbate hemolysis in falciparum malaria patients.

Characterization of the 3-HKT gene in important malaria vectors in India, viz: Anopheles culicifacies and Anopheles stephensi (Diptera: Culicidae)

The 3-hydroxykynurenine transaminase (3-HKT) gene plays a vital role in the development of malaria parasites by participating in the synthesis of xanthurenic acid, which is involved in the exflagellation of microgametocytes in the midgut of malaria vector species. The 3-HKT enzyme is involved in the tryptophan metabolism of Anophelines. The gene had been studied in the important global malaria vector, Anopheles gambiae. In this report, we have conducted a preliminary investigation to characterize this gene in the two important vector species of malaria in India, Anopheles culicifacies and Anopheles stephensi. The analysis of the genetic structure of this gene in these species revealed high homology with the An. gambiae gene. However, four non-synonymous mutations in An. stephensi and seven in An. culicifacies sequences were noted in the exons 1 and 2 of the gene; the implication of these mutations on enzyme structure remains to be explored.