Clinical and immunological profiles of anaemia in children and adolescents with Plasmodium vivax malaria in the Pará state, Brazilian Amazon.

Children and adolescents are at great risk for developing iron deficiency anaemia worldwide. In the tropical areas, malaria and intestinal parasites may also play an important role in anaemia pathogenesis. This study aimed at evaluating clinical and immunological aspects of anaemia in children and adolescents with Plasmodium vivax malaria, in the Pará State, Brazil. A longitudinal study was performed in two Reference Centers for malaria diagnosis in the Brazilian Amazon in children and adolescents with malaria (n = 81), as compared to a control group (n = 40). Patients had blood drawn three times [before treatment (D0), after treatment (D7) and at the first cure control (D30)] and hemogram, autoantibody analysis (anticardiolipin, antibodies against normal RBC membrane components) and cytokine studies (TNF and IL-10) were performed. Stool samples were collected for a parasitological examination. Malaria patients had a 2.7-fold greater chance of anaemia than the control group. At D0, 66.1% of the patients had mild anaemia, 30.5% had moderate and 3.5% had severe anaemia. Positivity to intestinal helminths and/or protozoa at stool examinations had no influence on anaemia. Patients had significantly lower levels of plasmatic TNF than control individuals at D0. Low TNF levels were more prevalent among patients with moderate/severe anaemia than in those with mild anaemia and among anaemic patients than in anaemic controls. TNF levels were positively correlated with the haemoglobin rates and negatively correlated with the interval time elapsed between the onset of symptoms and diagnosis. Both plasma TNF levels and haemoglobin rates increased during the follow-up period. The IL-10 levels were lower in patients than in the controls at day 0 and decreased thereafter up to the end of treatment. Only the anti-anticardiolipin autoantibodies were associated with moderate/severe anaemia and, possibly by reacting with the parasite glycosylphosphatidylinositol (a powerful stimulator of TNF production), may have indirectly contributed to decrease the TNF levels, which could be involved in the malarial vivax anaemia of these children and adolescents. More studies addressing this issue are necessary to confirm these findings and to add more information on the multifactorial pathogenesis of the malarial anaemia.

The impact of dark chocolate intake on arterial elasticity in individuals with HIV/AIDS undergoing ART: a randomized, double-blind, crossover trial.

An increase in the frequency of cardiovascular diseases has been observed in the HIV/AIDS population. Studies involving healthy subjects or subjects with other diseases have shown benefits of chocolate supplementation on endothelial function and vasodilation. We evaluate the impact of chocolate consumption on arterial elasticity in people living with human immunodeficiency virus - PLHIV. A double-blind, crossover trial including 110 PLHIV (19 to 59 years) on antiretroviral therapy - ART for at least 6 months and with a viral load of <500 copies per mL was conducted. All subjects were randomly assigned to 15-d dietary supplements containing dark chocolate or placebo with a 15-d washout period. Each participant received one of the two sequences: A (dark chocolate, placebo chocolate); B (placebo chocolate, dark chocolate). Arterial elasticity was measured using the HDI/PulseWave™ CR-2000 CardioVascular Profiling System®. Body composition, lipid profile, C-reactive protein, and thiobarbituric acid reactive substances were also assessed. Analysis of variance (ANOVA) for repeated measures using the Stata 11.0® program was used for cross-over analysis. Most subjects were men (59.0%) and Caucasian (46.1%) and the mean age was 44.6 ± 7.1 years. The mean time since diagnosis of HIV infection was 13.7 ± 5.3 years and the mean duration of ART was 12.9 ± 4.2 years. Chocolate consumption resulted in significant alterations in the large artery elasticity index - LAEI (p = 0.049) and the mean concentration of HDL-c was higher after supplementation with dark chocolate (p = 0.045). This is the first study to evaluate the effect of chocolate on arterial elasticity in PLHIV. The results showed that dark chocolate consumption for 15 days improved the elastic properties of the LAEI in PLHIV. These findings, added to the noninvasive method used, may expand the knowledge of CVDs in this population.

Multifocal electroretinography after high dose chloroquine therapy for malaria.

PURPOSE: To investigate changes in multifocal electroretinography (mfERG) parameters associated with high dose chloroquine therapy for treatment of malaria in the Amazonia region of Brazil. METHODS: Forty-eight subjects who had received chloroquine treatment for single or multiple malaria infections with a cumulative dose ranging from 1,050 to 27,000mg were included. The control group consisted of 37 healthy aged-matched subjects. Data was collected on amplitude and implicit time of the N1, P1 and N2 waves in the central macular hexagon (R1) and in five concentric rings at different retinal eccentricities (R2-R6). RESULTS: No significant difference was observed in any mfERG parameter between chloroquine treated patients and control subjects. A comparison with previous data obtained from patients with rheumatologic disorders in the same region of Brazil who had received larger cumulative doses of chloroquine and had displayed mfERG changes, indicated that retinal toxicity seems to be dependent on cumulative dose. CONCLUSION: Lack of mfERG changes in the current study suggests that intensive high dose chloroquine therapy for treatment of malaria is not associated with retinal toxicity.

Cellular and humoral immune responses against the Plasmodium vivax MSP-119 malaria vaccine candidate in individuals living in an endemic area in north-eastern Amazon region of Brazil.

BACKGROUND: Plasmodium vivax merozoite surface protein-1 (MSP-1) is an antigen considered to be one of the leading malaria vaccine candidates. PvMSP-1 is highly immunogenic and evidences suggest that it is target for protective immunity against asexual blood stages of malaria parasites. Thus, this study aims to evaluate the acquired cellular and antibody immune responses against PvMSP-1 in individuals naturally exposed to malaria infections in a malaria-endemic area in the north-eastern Amazon region of Brazil. METHODS: The study was carried out in Paragominas, Pará State, in the Brazilian Amazon. Blood samples were collected from 35 individuals with uncomplicated malaria. Peripheral blood mononuclear cells were isolated and the cellular proliferation and activation was analysed in presence of 19 kDa fragment of MSP-1 (PvMSP-119) and Plasmodium falciparum PSS1 crude antigen. Antibodies IgE, IgM, IgG and IgG subclass and the levels of TNF, IFN-γ and IL-10 were measured by enzyme-linked immunosorbent assay. RESULTS: The prevalence of activated CD4+ was greater than CD8+ T cells, in both ex-vivo and in 96 h culture in presence of PvMSP-119 and PSS1 antigen. A low proliferative response against PvMSP-119 and PSS1 crude antigen after 96 h culture was observed. High plasmatic levels of IFN-γ and IL-10 as well as lower TNF levels were also detected in malaria patients. However, in the 96 h supernatant culture, the dynamics of cytokine responses differed from those depicted on plasma assays; in presence of PvMSP-119 stimulus, higher levels of TNF were noted in supernatant 96 h culture of malaria patient's cells while low levels of IFN-γ and IL-10 were verified. High frequency of malaria patients presenting antibodies against PvMSP-119 was evidenced, regardless class or IgG subclass.PvMSP-119-induced antibodies were predominantly on non-cytophilic subclasses. CONCLUSIONS: The results presented here shows that PvMSP-119 was able to induce a high cellular activation, leading to production of TNF and emphasizes the high immunogenicity of PvMSP-119 in naturally exposed individuals and, therefore, its potential as a malaria vaccine candidate.

Mercury exposure, serum antinuclear/antinucleolar antibodies, and serum cytokine levels in mining populations in Amazonian Brazil: a cross-sectional study.

Mercury is an immunotoxic substance that has been shown to induce autoimmune disease in rodent models, characterized by lymphoproliferation, overproduction of immunoglobulin (IgG and IgE), and high circulating levels of auto-antibodies directed at antigens located in the nucleus (antinuclear auto-antibodies, or ANA) or the nucleolus (antinucleolar auto-antibodies, or ANoA). We have reported elevated levels of ANA and ANoA in human populations exposed to mercury in artisanal gold mining, though other confounding variables that may also modulate ANA/ANoA levels were not well controlled. The goal of this study is to specifically test whether occupational and environmental conditions (other than mercury exposure) that are associated with artisanal gold mining affect the prevalence of markers of autoimmune dysfunction. We measured ANA, ANoA, and cytokine concentrations in serum and compared results from mercury-exposed artisanal gold miners to those from diamond and emerald miners working under similar conditions and with similar socio-economic status and risks of infectious disease. Mercury-exposed gold miners had higher prevalence of detectable ANA and ANoA and higher titers of ANA and ANoA as compared to diamond and emerald miners with no occupational mercury exposure. Also, mercury-exposed gold miners with detectable ANA or ANoA in serum had significantly higher concentrations of pro-inflammatory cytokines IL-1beta, TNF-alpha, and IFN-gamma in serum as compared to the diamond and emerald miners. This study provides further evidence that mercury exposure may lead to autoimmune dysfunction and systemic inflammation in affected populations.

The influence of birthweight on arterial blood pressure of children.

BACKGROUND & AIMS: This study examined the relationship between birthweight and blood pressure in childhood. METHODS: Prospective cohort study involving 472 Brazilian children ranging in age from 5 to 8 years. Birthweight, systolic blood pressure (SBP), diastolic blood pressure (DBP), body mass index (BMI), total cholesterol and fractions (LDL-c, HDL-c), and triglycerides were determined. Total cholesterol, LDL-c, HDL-c, and triglycerides were assessed by automated enzymatic methods. Blood pressure was measured with the HDI/Pulse Wave CR-2000 equipment. Multiple regression models were used to investigate the relationship between birthweight and SBP and DBP, controlling for the following variables: gender, age, BMI, total cholesterol, triglycerides, per capita income, and maternal education. RESULTS: When adjusting for gender and BMI, we found a systolic blood pressure increase of 2.9 (95% CI=-5.33 to -0.56) mmHg per kilogram birthweight reduction. The unadjusted association was insignificant. CONCLUSION: Our data suggest that low birthweight is one of the factors contributing to blood pressure elevation at early ages. A way to prevent these diseases is by implementing public policies focused on good nutrition and adequate prenatal care for pregnant women.

Chloroquine and sulphadoxine-pyrimethamine sensitivity of Plasmodium falciparum parasites in a Brazilian endemic area.

BACKGROUND: The goal of the present study was the characterization of Plasmodium falciparum genes associated to malaria drug resistance (pfcrt, pfdhfr and pfdhps), in samples from two Brazilian localities. METHODS: Parasites from 65 P. falciparum samples were genotyped using nested-PCR and direct DNA sequencing. RESULTS: Six resistant sulphadoxine-pyrimethamine (SP) pfdhfr genotypes and one haplotype associated to SP sensitivity were detected. For pfcrt gene, SVMNT chloroquine (CQ)-resistant genotype was detected as well as the CVMNK CQ-sensitive haplotype in the same sample from Paragominas, that showed a SP-sensitive genotype. CONCLUSION: This study is the first to document the sensitivity of P. falciparum parasites to CQ and SP in Brazilian field samples. The importance of these findings is discussed.

Analysis of genetic variability of Plasmodium vivax isolates from different Brazilian Amazon areas using tandem repeats.

Few genetic markers have been described to analyze populations of Plasmodium vivax. The genetic variability of P. vivax has been analyzed mainly among isolates taken from areas ranging from hyper- to holoendemic areas. These studies of genetic variability have neglected many areas with different epidemiologic profiles. The purpose of this study was to analyze the genetic variability of P. vivax isolates from four different Brazilian Amazon areas. We chose to study the five most polymorphic tandem repeats (TRs) identified so far. All TRs studied were polymorphic in at least one studied population, with a modal allele at nearly all loci. Expected heterozygosity ranged from 0.462 to 0.666 and did not correlate with the repeat array length. The genetic distances among the populations varied from 0.027 to 0.241, and did not correlate with their geographic separation. Tandem repeats identified in P. vivax isolates failed to allow geographic clustering.

Real-time PCR versus conventional PCR for malaria parasite detection in low-grade parasitemia.

We have optimized a faster and cheaper real-time PCR and developed a conventional genus specific PCR based on 18S rRNA gene to detect malaria parasites in low-grade parasitemias. Additionally, we compared these PCRs to the OptiMAL-IT test. Since there is no consensus on choice of standard quantitative curve in real-time assays, we decided to investigate the performance of parasite DNA from three different sources: "genome", amplicon and plasmid. The amplicon curve showed the best efficiency in quantifying parasites. Both PCR assays detected 100% of the clinical samples tested; the sensitivity threshold was 0.5 parasite/mul and no PCR positive reaction occurred when malaria parasites were not present. Conversely, if OptiMAL-IT were employed for malaria diagnosis, 30% of false-negative results could be expected. We conclude that PCR assays have potential for detecting malaria parasites in asymptomatic infections, in evaluation of malaria vaccine molecule candidates, for screening blood donors, especially in endemic areas, or even in monitoring malaria therapy.

Some aspects of the behavior of the hypothalamus-pituitary-adrenal axis in patients with uncomplicated Plasmodium falciparum malaria: Cortisol and dehydroepiandrosterone levels.

We studied the behavior of cortisol and dehydroepiandrosterone (DHEA) in 24 patients with uncomplicated Plasmodium falciparum malaria of the Evandro Chagas Institute, Belém, Pará, Brazil. The patients were evaluated before treatment (Day 0), 24h after the beginning of medication (Day 1) and on Day 8 of follow-up (Day 7). Steroid levels were correlated with parasitemia, temperature and time of the disease. The levels of these hormones were found to be significantly higher on Day 0 than on Day 7, showing no correlation with parasitemia or temperature, but temperature had a positive effect on the correlation between cortisol and dehydroepiandrosterone. Cortisol was not correlated with the time of disease, but a significant negative correlation was observed between DHEA and time of disease on Day 7, suggesting a decline in the adrenal reserve of this steroid. In conclusion, an increase in cortisol and dehydroepiandrosterone is observed in patients with falciparum malaria, with these levels declining with decreasing parasitemia. The finding that temperature interfered with the correlation between cortisol and dehydroepiandrosterone suggests a common mechanism for the activation of these hormones in malaria.

[Hemoglobin concentration in children from birth to one year of age]. / Concentração de hemoglobina em crianças do nascimento até um ano de vida.

This study aimed to describe the evolution of hemoglobin concentration considering the following factors: birth weight, growth rate, and food intake. The cross-sectional study focused on infants (<1 year of age) whose growth and development were monitored by public health services in cities located in the five geographic regions of Brazil. Some 51.7% of the children aged 6 to 12 months presented anemia. Estimation of [Hb] concentration by a theoretical equation suggested that endogenous iron is able to maintain normal Hb levels in the first three months of life. Prevalence of reduced Hb was higher in low birth weight infants. Growth rate, verified by the difference between present weight and birth weight in Z scores, was not the relevant factor for hemoglobin concentration. The relevance of breastfeeding was evident in the initial months of life. However, complementary foods did not influence infant Hb concentration. The high prevalence of low Hb levels calls attention to the need for programs to control anemia.

[Risks associated with the weaning process in children born in a university hospital: a prospective cohort in the first year of life, São Paulo, 1998-1999]. / Riscos associados ao processo de desmame entre crianças nascidas em hospital universitário de São Paulo, entre 1998 e 1999: estudo de coorte prospectivo do primeiro ano de vida.

This study describes breastfeeding practices with children born in a university hospital in the city of S o Paulo, Brazil, and identifies factors associated with duration of breastfeeding and exclusive breastfeeding. A cohort of 506 children was identified; of these, it was possible to analyze information on feeding practices for 450 infants at least until the second month of life. Daily information on infant feeding was recorded by mothers in a food frequency questionnaire. Survival analysis techniques (Kaplan-Meier and Cox) were used. Median duration of breastfeeding and exclusive breastfeeding were 205 and 23 days, respectively. The principal factors related to exclusive breastfeeding were mother's age (hr young/old = 1.22; 95% CI = 1.006-1.486) and mother's schooling (hr primary/university = 2.13; 95% CI = 1.381-3.307 and hr secondary/university = 1.78; 95% CI = 1.145-2.792), which could be related to knowledge concerning the benefits of exclusive breastfeeding.

[Importance of clinical and laboratory profiles for the differential diagnosis of malaria and acute viral hepatitis]. / A importância do perfil clínico-laboratorial no diagnóstico diferencial entre malária e hepatite aguda viral.

OBJECTIVE: To establish clinical and diagnostic findings of malaria and acute viral hepatitis in children, stressing similarities and differences, so as to enhance the sensitivity of early malaria diagnosis in childhood. METHODS: Two groups were studied, each including 30 children between 2 and 10 years of age. The patients presented either primary malaria infection or acute viral hepatitis, confirmed by thick blood film and tests for markers of viral hepatitis A and B. The patients were submitted to the following evaluations: erythrocyte, leucocyte and platelet counts, hemoglobin and hematocrit dosage, hepatic enzymes, urea, creatinine and bilirubin dosage. Clinical and laboratory findings were described for both groups and compared. Individuals with alterations on the physical exam in both groups were compared using Fisher's exact test. RESULTS: Baseline clinical findings were the same in all patients: fever, headache, digestive problems and dark urine. One half of malaria patients did not present the classical malaria signs, but all of them presented fever, differently from patients with hepatitis. In malaria patients, anemia and thrombocytopenia were significantly more frequent than in hepatitis patients. A remarkable increase of bilirubin and hepatic enzyme levels was found in hepatitis patients. CONCLUSIONS: A detailed physical examination and a thorough evaluation of non-specific laboratory tests are sufficient to allow the presumptive diagnosis of both malaria and viral hepatitis, and to reinforce the early diagnosis and treatment of malaria.

[Short course schemes for vivax malaria treatment]. / Esquemas terapêuticos encurtados para o tratamento de malária por Plasmodium vivax.

with the objective of evaluating shortened therapeutic outlines effective in vivax malaria treatment, we accomplished an open, prospective study allocating 234 patients with vivax malaria distributed at random into eight therapeutic groups. Six groups used oral arthemisin as blood esquizonticide at different doses for one day and the other two groups received chloroquine in a single dose. The primaquine was used as a hypnozoiticide in all groups. They received a daily dose of 30mg in the course of five or seven days in all groups. The clearance of parasitaemia in patients treated with arthemisin (independent of dosage) was faster than the chloroquine group (p <0.01). Cure was acheived in 92.3% and 80.2%, in patients treated with primaquine for seven or five days, respectively (p=0.0372).

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.

Assessment of mercury exposure and malaria in a Brazilian Amazon riverine community.

Small-scale gold mining in the Brazilian Amazon occurs in areas with high rates of malaria transmission. Amazonian populations can be exposed to mercury through direct contact with the mining process and/or through fish consumption. Because of data from experimental studies, we examined the potential for mercury to affect host response to malaria. A cross-sectional survey was done in Jacareacanga, a riverine community in Para state, in a region of intense alluvial gold mining. A sample of 205 persons was selected by cluster sampling from the total population of approximately 2000. A brief medical history and exam were conducted, malaria slides were obtained, and hair samples were taken to measure mercury levels. The average hair mercury level was 8.6 micrograms/g, ranging from 0.3 to 83.2 micrograms/g. The most important predictors of elevated mercury levels were high fish consumption and low income. Although there was no prevalent malaria, the odds of reporting a past malaria infection was four times higher for those also reporting a history of working with mercury.

Mercury exposure and malaria prevalence among gold miners in Pará, Brazil.

Economic development, including resource extraction, can cause toxic exposures that interact with endemic infectious diseases. Mercury is an immunotoxic metal used in the amalgamation of gold, resulting in both occupational exposures and environmental pollution. A cross-sectional medical survey was conducted in 1997 on 135 garimpeiros in Para, Brazil, because of their risks of both mercury exposure and malaria transmission. Mean levels of blood and urine mercury were well above non-exposed background levels. Twenty-six subjects had malaria parasitemia: Health symptoms consistent with mercury exposure were reported, but neither symptoms nor signs correlated with mercury levels in blood or urine. We did not find a dose response relationship between mercury exposure and likelihood of prevalent malaria infection, but there was a possible reduction in acquisition of immunity that may be associated with conditions in gold mining, including mercury exposure.