Maria das Graças Costa Alecrim

Severe Plasmodium vivax Malaria, Brazilian Amazon

We describe a case series of 17 patients hospitalized in Manaus (western Brazilian Amazon) with PCR-confirmed Plasmodium vivax infection who were treated with chloroquine and primaquine. The major complications were jaundice and severe anemia. No in vivo chloroquine resistance was detected. These data help characterize the clinical profile of severe P. vivax malaria in Latin America.

Chloroquine-Resistant Plasmodium vivax, Brazilian Amazon

To the Editor: Plasmodium vivax is the protozoan that causes the second most common form of malaria. Some resistant strains to chloroquine (CQ) occur in a few places in Asia and the Indo-Pacific Region (1–4). Although resistance of P. vivax to CQ has already been described in South America (5–7), there are limited data regarding this issue. CQ plus primaquine is the standard treatment for vivax malaria worldwide. Presently, this drug regimen exhibits satisfactory efficacy in the Brazilian Amazon. However, in recent years several treatment failures presumably related to CQ resistance, have been reported in the city of Manaus (Amazonas) where vivax malaria predominates (7). This observation warrants local attention despite these cases having no confirmation of CQ blood levels on the basis of the appearance of asexual parasites against CQ plus desethylchloroquine levels exceeding the minimally effective plasma concentration proposed for sensitive parasite strains (>10 ng/mL) (8), according to Pan American Health Organization recommendations (9). From September 2004 to February 2005, a 28-day in vivo test was conducted at the Foundation for Tropical Medicine of Amazonas (FMTAM) in Manaus, Brazil, to assess the efficacy of standard supervised CQ therapy. The test involved 166 volunteers with uncomplicated vivax malaria. Each volunteer was administered uncoated, scored, 150-mg CQ tablets (10 + 7.5 + 7.5 mg/kg at 24-hour intervals) (9). Primaquine was withheld until day 28 (dose regimen of 30 mg/day for 7 days). Among the 109 volunteers who completed the in vivo test, 19 had positive blood smears within the 28-day follow-up (1 on day 14, 3 on day 21, and 15 on day 28). All were required to undergo alternative therapy (mefloquine). Adequate CQ absorption was confirmed in these cases on day 2 with a mean ± SD CQ plasma concentration of 785.4 ± 800.1 ng/mL) (10) Suspected therapeutic failure (P. vivax CQ resistance) was confirmed in 11 (10.1%) of 109 persons with a mean isolated choloroquine plasma concentration >10 ng/mL (356.6 ± 296.1 ng/mL) (9). Desethylchloroquine levels in plasma were not measured. Previously, a CQ efficacy study demonstrated that 4.4% of those tested had CQ-resistant P. vivax (7). In comparison, the proportion of failures (10.1%) in the current study seems to be relevant; even though most of the P. vivax infections (98, 89.9%) were successfully evaluated and adequate clinical and parasitologic responses were obtained. Currently, the FMTAM Manaus Outpatient Clinic is detecting patients from different areas of the city who show parasitologic recurrences after correct treatment within 28 days of the routine clinical follow-up. This observation is an indirect indicator of the possible regional spread of P. vivax CQ-resistant strains (unpub. data). We believe our findings are important and merit the attention of local public health authorities. Considering the possibility of emerging underestimated P. vivax CQ resistance in Manaus, we feel it is essential to quickly clarify whether such documented resistance can copromote vivax malaria outbreaks in malaria-endemic areas within the Amazon.

Plasmodium vivax resistance to chloroquine (R2) and mefloquine (R3) in Brazilian Amazon region

We report for the first time a patient with malaria due to Plasmodium vivax who showed R2 resistance to chloroquine and R3 resistance to mefloquine in the Brazilian Amazon region based on WHO clinical criteria for diagnosis of malaria resistance. Failure was observed with unsupervised oral chloroquine, chloroquine under rigorous supervision and mefloquine in the same scheme. Finally, the patient was cured with oral artesunate.

pfcrt Polymorphism and the Spread of Chloroquine Resistance in Plasmodium falciparum Populations across the Amazon Basin

The widespread occurrence of drug-resistant malaria parasites in South America presents a formidable obstacle to disease control in this region. To characterize parasite populations and the chloroquine-resistance profile of Plasmodium falciparum in the Amazon Basin, we analyzed a DNA segment of the pfcrt gene, spanning codons 72-76, and genotyped 15 microsatellite (MS) markers in 98 isolates from 6 areas of Brazil, Peru, and Colombia where malaria is endemic. The K76T mutation, which is critical for chloroquine resistance, was found in all isolates. Five pfcrt haplotypes (S[tct]MNT, S[agt]MNT, CMNT, CMET, and CIET) were observed, including 1 previously found in Asian/African isolates. MS genotyping showed relatively homogeneous genetic backgrounds among the isolates, with an average of 3.8 alleles per marker. Isolates with identical 15-loci MS haplotypes were found in different locations, suggesting relatively free gene flow across the Amazon Basin. Allopatric isolates carrying SMNT and CMNT haplotypes have similar genetic backgrounds, although parasites carrying the CIET haplotype have some exclusive MS alleles, suggesting that parasites with CIET alleles were likely to have been introduced into Brazil from Asia or Africa. This study provides the first evidence of the Asian pfcrt allele in Brazil and a detailed analysis of P. falciparum populations, with respect to pfcrt haplotypes, in the Amazon Basin.

Concurrent Helminthic Infection Protects Schoolchildren with Plasmodium vivax from Anemia

Background Plasmodium vivax is responsible for a significant portion of malaria cases worldwide, especially in Asia and Latin America, where geo-helminthiasis have a high prevalence. Impact of the interaction between vivax malaria and intestinal helminthes has been poorly explored. The objective of this study was to evaluate the influence of intestinal helminthiasis on the concentration of hemoglobin in children with Plasmodium vivax malaria in rural areas in the municipality of Careiro, in the Western Brazilian Amazon. Methodology/Principal Findings A cohort study was conducted from April to November 2008, enrolling children from 5 to 14 years old in two rural areas endemic for malaria. A cross-sectional evaluation was performed in April to actively detect cases of malaria and document baseline hemoglobin and nutritional status. Children were followed-up for six months through passive case detection of malaria based on light microscopy. Throughout the follow-up interval, hemoglobin value and stool examination (three samples on alternate days) were performed on children who developed P. vivax malaria. For 54 schoolchildren with a single infection by P. vivax, hemoglobin during the malaria episode was similar to the baseline hemoglobin for children co-infected with Ascaris lumbricoides (n = 18), hookworm (n = 11) and Trichuris trichiura (n = 9). In children without intestinal helminthes, a significant decrease in the hemoglobin during the malarial attack was seen as compared to the baseline concentration. In the survival analysis, no difference was seen in the time (in days) from the baseline cross-sectional to the first malarial infection, between parasitized and non-parasitized children. Conclusion/Significance For the first time, a cohort study showed that intestinal helminthes protect against hemoglobin decrease during an acute malarial attack by P. vivax.

Pattern of humoral immune response to Plasmodium falciparum blood stages in individuals presenting different clinical expressions of malaria

BackgroundThe development of protective immunity against malaria is slow and to be maintained, it requires exposure to multiple antigenic variants of malaria parasites and age-associated maturation of the immune system. Evidence that the protective immunity is associated with different classes and subclasses of antibodies reveals the importance of considering the quality of the response. In this study, we have evaluated the humoral immune response against Plasmodium falciparum blood stages of individuals naturally exposed to malaria who live in endemic areas of Brazil in order to assess the prevalence of different specific isotypes and their association with different malaria clinical expressions.MethodsDifferent isotypes against P. falciparum blood stages, IgG, IgG1, IgG2, IgG3, IgG4, IgM, IgE and IgA, were determined by ELISA. The results were based on the analysis of different clinical expressions of malaria (complicated, uncomplicated and asymptomatic) and factors related to prior malaria exposure such as age and the number of previous clinical malaria attacks. The occurrence of the H131 polymorphism of the FcγIIA receptor was also investigated in part of the studied population.ResultsThe highest levels of IgG, IgG1, IgG2 and IgG3 antibodies were observed in individuals with asymptomatic and uncomplicated malaria, while highest levels of IgG4, IgE and IgM antibodies were predominant among individuals with complicated malaria. Individuals reporting more than five previous clinical malaria attacks presented a predominance of IgG1, IgG2 and IgG3 antibodies, while IgM, IgA and IgE antibodies predominated among individuals reporting five or less previous clinical malaria attacks. Among individuals with uncomplicated and asymptomatic malaria, there was a predominance of high-avidity IgG, IgG1, IgG2 antibodies and low-avidity IgG3 antibodies. The H131 polymorphism was found in 44.4% of the individuals, and the highest IgG2 levels were observed among asymptomatic individuals with this allele, suggesting the protective role of IgG2 in this population.ConclusionTogether, the results suggest a differential regulation in the anti-P. falciparum antibody pattern in different clinical expressions of malaria and showed that even in unstable transmission areas, protective immunity against malaria can be observed, when the appropriated antibodies are produced.

Analysis of Single-Nucleotide Polymorphisms in the crt-o and mdr1 Genes of Plasmodium vivax among Chloroquine-Resistant Isolates from the Brazilian Amazon Region

ABSTRACT Plasmodium vivax parasites with chloroquine resistance (CQR) are already circulating in the Brazilian Amazon. Complete single-nucleotide polymorphism (SNP) analyses of coding and noncoding sequences of the pvmdr1 and pvcrt-o genes revealed no associations with CQR, even if some mutations had not been randomly selected. In addition, striking differences in the topologies and numbers of SNPs in these transporter genes between P. vivax and P. falciparum reinforce the idea that mechanisms other than mutations may explain this virulent phenotype in P. vivax.

Geographic Structuring of the Plasmodium falciparum Sarco(endo)plasmic Reticulum Ca2+ ATPase (PfSERCA) Gene Diversity

Artemisinin, a thapsigargin-like sesquiterpene has been shown to inhibit the Plasmodium falciparum sarco/endoplasmic reticulum calcium-ATPase PfSERCA. To collect baseline pfserca sequence information before field deployment of Artemisinin-based Combination therapies that may select mutant parasites, we conducted a sequence analysis of 100 isolates from multiple sites in Africa, Asia and South America. Coding sequence diversity was large, with 29 mutated codons, including 32 SNPs (average of one SNP/115 bp), of which 19 were novel mutations. Most SNP detected in this study were clustered within a region in the cytosolic head of the protein. The PfSERCA functional domains were very well conserved, with non synonymous mutations located outside the functional domains, except for the S769N mutation associated in French Guiana with elevated IC50 for artemether. The S769N mutation is located close to the hinge of the headpiece, which in other species modulates calcium affinity and in consequence efficacy of inhibitors, possibly linking calcium homeostasis to drug resistance. Genetic diversity was highest in Senegal, Brazil and French Guiana, and few mutations were identified in Asia. Population genetic analysis was conducted for a partial fragment of the gene encompassing nucleotide coordinates 87-2862 (unambiguous sequence available for 96 isolates). This supported a geographic clustering, with a separation between Old and New World samples and one dominant ancestral haplotype. Genetic drift alone cannot explain the observed polymorphism, suggesting that other evolutionary mechanisms are operating. One possible contributor could be the frequency of haemoglobinopathies that are associated with calcium dysregulation in the erythrocyte.

Glucose-6-phosphate dehydrogenase deficient variants are associated with reduced susceptibility to malaria in the Brazilian Amazon

BACKGROUND Glucose-6-phosphate dehydrogenase deficiency (G6PDd) has been shown to protect against malaria infection and severe manifestations in African and Asia, but there is a scarcity of studies in the Americas. This study aimed to study the prevalence of G6PDd and its association with malaria occurrence in the Brazilian Amazon. METHODS A cross-sectional study was conducted in the male population to estimate the prevalence of G6PDd and malaria infection. G6PD deficient samples were genotyped to identify the deficient variant. Number of previous malaria episodes and need for blood transfusion during malaria episodes were recorded by applying a standardized questionary. RESULTS From a sample of 1478 male individuals, 66 were detected as G6PD deficient, resulting in a prevalence of of 4.5% (95% CI = 3.44-5.56%). Fifty six G6PD deficient individuals (3.8%; 95% CI = 2.82-4.77) presented the G6PD A-variant mutation, while 10 individuals (0.7%; 95% CI = 0.42-0.97) severely deficient were genotyped as carriers of the G6PD Mediterranean variant. After adjusting for age, G6PD deficient individuals were less likely to report the occurrence of malaria episodes, and the protective effect was related to the enzyme activity, with carriers of the GG6PD A-variant presenting a 88% reduction (AOR: 0.119; 95% CI = 0.057-0.252; p < 0.001) and carriers of the Meditarrenean variant presenting 99% lower risk (AOR: 0.010; 95% CI = 0.002-0.252; p < 0.001) when compared to non-deficient individuals. On the other hand, G6PD deficient subjects reported higher need of transfusion during malaria episodes (p < 0.001). CONCLUSION G6PD enzyme activity was directly related to susceptibility to malaria in the Brazilian Amazon, where P. vivax predominates. Severe G6PDd was associated with considerable higher risk of malaria-related transfusions.

Glucose-6-phosphate dehydrogenase deficiency in an endemic area for malaria in Manaus: a cross-sectional survey in the Brazilian Amazon.

Background There is a paucity of information regarding glucose-6-phosphate dehydrogenase (G6PD) deficiency in endemic areas for malaria in Latin America. Methodology/Principal Findings This study determined the prevalence of the G6PD deficiency in 200 male non-consanguineous individuals residing in the Ismail Aziz Community, on the outskirts of Manaus (Brazilian Amazon). Six individuals (3%) were deficient using the qualitative Brewers test. Gel electrophoresis showed that five of these patients were G6PD A−. The deficiency was not associated with the ethnic origin (P = 0.571). In a multivariate logistic regression analysis, G6PD deficiency protected against three or more episodes of malaria (P = 0.049), independently of the age, and was associated with a history of jaundice (P = 0.020) and need of blood transfusion (P = 0.045) during previous treatment for malarial infection, independently of the age and the previous malarial exposure. Conclusions/Significance The frequency of G6PD deficiency was similar to other studies performed in Brazil and the finding of a predominant G6PD A− variant will help the clinical management of patients with drug-induced haemolysis. The history of jaundice and blood transfusion during previous malarial infection may trigger the screening of patients for G6PD deficiency. The apparent protection against multiple malarial infections in an area primarily endemic for Plasmodium vivax needs further investigation.