Ricardo Machado

Genetic Structure of Plasmodium falciparum Populations in the Brazilian Amazon Region

After a major increase in incidence between the 1970s and the 1990s, the Brazilian Amazon region now accounts for the most cases of Plasmodium falciparum malaria in the Americas. Polymorphism of 10 microsatellite loci in the P. falciparum genome was studied in 196 isolates obtained from 5 populations in the region. There was significant multilocus linkage disequilibrium, particularly within populations with lower proportions of mixed-genotype infections. However, most multilocus genotypes in different isolates were distinct, and there was no evidence of any recent epidemic expansion of particular clones. Genetic divergence between populations was very substantial but did not fit a simple model of isolation by distance. Thus, different foci of P. falciparum in Brazil are quite independent, with distinct population structures and minimal gene flow, a finding that has implications for strategies to control infection and to contain the spread of drug resistance at a regional level.

Extreme geographical fixation of variation in the Plasmodium falciparum gamete surface protein gene Pfs48/45 compared with microsatellite loci.

Comparing patterns of genetic variation at multiple loci in the genome of a species can potentially identify loci which are under selection. The large number of polymorphic microsatellites in the malaria parasite Plasmodium falciparum are available markers to screen for selectively important loci. The Pfs48/45 gene on Chromosome 13 encodes an antigenic protein located on the surface of parasite gametes, which is a candidate for a transmission blocking vaccine. Here, genotypic data from 255 P. falciparum isolates are presented, which show that alleles and haplotypes of five single nucleotide polymorphisms (SNPs) in the Pfs48/45 gene are exceptionally skewed in frequency among different P. falciparum populations, compared with alleles at 11 microsatellite loci sampled widely from the parasite genome. Fixation indices measuring inter-population variance in allele frequencies (F(ST)) were in the order of four to seven times higher for Pfs48/45 than for the microsatellites, whether considered (i) among populations within Africa, or (ii) among different continents. Differing mutational processes at microsatellite and SNP loci could generally affect the population structure at these different types of loci, to an unknown extent which deserves further investigation. The highly contrasting population structure may also suggest divergent selection on the amino acid sequence of Pfs48/45 in different populations, which plausibly indicates a role for the protein in determining gamete recognition and compatibility.

Distribution of Plasmodium vivax variants (VK210, VK247 and P. vivax-like) in three endemic areas of the Amazon region of Brazil and their correlation with chloroquine treatment

The present study evaluated the glass fibre membrane (GFM)-polymerase chain reaction (PCR)-enzyme-linked immunosorbent assay (ELISA) technique for genotyping the Plasmodium vivax variants, to verify the distribution of P. vivax variants (VK210, VK247 and P. vivax-like) in parts of Brazil and their correlation with levels of parasitaemia, previous malaria experience and clearance of parasitaemia linked to different treatment schedules. The samples were taken from individuals living in Macapá, Porto Velho and Belém, all of which are endemic areas of vivax malaria in the Amazon region of Brazil. Blood samples were collected on GFMs. The gene that codes for the circumsporozoite proteins of P. vivax variants was amplified by PCR and the amplified fragments were hybridized to variant-specific, digoxigenin-labelled oligonucleotide probes by ELISA. The GFM-PCR-ELISA technique was shown to be accurate for epidemiological surveys of the vivax complex. All variants were detected in all 3 areas, but only P. vivax VK210 was found as a single agent of infection, while the other 2 occurred as mixed infections. The P. vivax-like variant was found to be associated with low parasitaemia and VK210 with the highest parasitaemia levels; none of the P. vivax variants was linked with a previous malaria experience. In all cases parasitaemia clearance was identical regarding the type of treatment and consequently it is not possible to confirm the previously reported correlation between P. vivax genotype and response to chloroquine.

Susceptibility of Anopheles aquasalis and An. darlingi to Plasmodium vivax VK210 and VK247

The susceptibility of Anopheles aquasalis (F3 generation) and An. darlingi (F1 generation) to Plasmodium vivax circumsporozoite protein phenotypes from a limited number of blood samples of malaria patients in Belém, state of Pará, Brazil, was examined A polymerase chain reaction was used to determine the P. vivax phenotypes in blood samples and the blood-fed infected mosquitoes were dissected and tested by ELISA. In all patient infections, more infected An. aquasalis and An. darlingi were positive for VK210 compared with VK247.

Concurrent dengue and malaria in the Amazon region

INTRODUCTION The Amazon region has extensive forested areas and natural ecosystems, providing favorable conditions for the existence of innumerous arboviruses. Over 200 arboviruses have been isolated in Brazil and about 40 are associated with human disease. Four out of 40 are considered to be of public health importance in Brazil: Dengue viruses (1-4), Oropouche, Mayaro and Yellow Fever. Along with these viruses, about 98% of the malaria cases are restricted to the Legal Amazon region. METHODS This study aimed to investigate the presence of arboviruses in 111 clinical serum samples from patients living in Novo Repartimento (Pará), Plácido de Castro (Acre), Porto Velho (Rondônia) and Oiapoque (Amapá). The viral RNA was extracted and RT-PCR was performed followed by a Multiplex-Nested-PCR, using Flavivirus, Alphavirus and Orthobunyavirus generic and species-specific primers. RESULTS Dengue virus serotype 2 was detected in two patients living in Novo Repartimento (Pará) that also presented active Plasmodium vivax infection. CONCLUSIONS Despite scant data, this situation is likely to occur more frequently than detected in the Amazon region. Finally, it is important to remember that both diseases have similar clinical findings, thus the diagnosis could be made concomitantly for dengue and malaria in patients living or returning from areas where both diseases are endemic or during dengue outbreaks.

Comparação de quatro métodos laboratoriais para diagnóstico da Giardia lamblia em fezes de crianças residentes em Belém, Pará

We report the evaluation of four techniques for Giardia lamblia diagnosis in childrens stool. The Iron haematoxilin staining and direct examination with lugol showed lower positivity, while the method of Faust et al. Continues to be a good option for G. lamblia diagnosis and Immunoenzymatic assay increases the detection of this parasite.We report the evaluation of four techniques for Giardia lamblia diagnosis in childrens stool. The Iron haematoxilin staining and direct examination with lugol showed lower positivity, while the method of Faust et al. Continues to be a good option for G. lamblia diagnosis and Immunoenzymatic assay increases the detection of this parasite.

Correlation between Plasmodium vivax variants in Belém, Pará State, Brazil and symptoms and clearance of parasitaemia

The aim of this study was to determine how different types of P. vivax affect clinical symptoms and parasitaemia clearance. Blood was collected from individuals from Pará State, Brazil. The patients were treated as chloroquine plus primaquine. P. vivax were typed daily till D7 and again on D30. Now we can confirm a previously reported correlation between P. vivax genotype and response to chloroquine. Clinical symptoms do not allow for objective identification of different P. vivax types in the Brazilian Amazon, since the VK247 and P. vivax-like have only been detected in mixed infections.

Etiological agents of diarrhea in patients infected by the human immunodeficiency virus-1: a review

Despite the importance of understanding the epidemiology of agents responsible for infectious diarrhea in human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) population, the number of articles about this subject is relatively few. The current article summarizes published data on bacterial, fungal, viral and parasitic enteropathogens in the HIV/AIDS seropositive subjects in different countries, regions and localities. In general, there is a great difference in the frequencies of etiological agents due to factors which include immune status, geographical location, climate and socioeconomic conditions. It is important to stress that a great prevalence of infection by emergent agents has been reported in the more advanced stages of AIDS. Therefore, to establish specific treatment depends directly on knowledge of these agents and risk factors associated to their distribution. Moreover, the colonization by potential pathogenic agents verified in these individuals is high thus implicating that they act as carriers. Finally, public health measures of control and prevention must take into consideration the regional previously identified enteropathogens, especially in areas where HIV prevalence is high.

Temporal and spatial distribution of the variants of merozoite surface protein-1 (MSP-1) in Plasmodium falciparum populations in Brazil.

The polymorphic, merozoite surface protein-1 (MSP-1) of Plasmodium falciparum, an antigen of the parasites asexual blood-stages, is a major malaria-vaccine candidate. Nucleotide sequences of each variable domain or block of this antigen may be grouped into one of three possible allelic types (K1, MAD20 and RO33), and 24 major types of the msp-1 gene may be defined, as unique combinations of allelic types in these variable blocks. Isolates collected from the Brazilian Amazon, over a period of 14 years, have now been investigated, by PCR-based typing of the msp-1 gene. Thirteen of the 24 possible gene-types were identified, and 336 P. falciparum clones were fully typed among 239 isolates. Most parasites (87%) belonged to one of the seven most frequent gene-types. Marked temporal variation in the distribution of msp-1 variants was found when comparing parasites sampled in the same sites at intervals of at least 5 years. Spatial variations were also found when comparing parasites from both neighbouring and distant sites within the Amazon Basin. The between-population variance in the frequencies of msp-1 allelic types found in Brazil, as estimated by Wrights FST statistic, is of similar magnitude to that found in previous world-wide comparisons. The potential implications of these findings for the development of an MSP-1-based, multivalent malaria vaccine are discussed.

Plasmodium vivax circumsporozoite variants and Duffy blood group genotypes in the Brazilian Amazon region

The circumsporozoite protein (CSP) of the Plasmodium vivax infective sporozoite is considered to be a major target for the development of recombinant malaria vaccines. The Duffy blood group molecule acts as the red blood cell receptor for P. vivax. We review the frequency of P. vivax CSP variants and report their association with the Duffy blood group genotypes from Brazilian Amazon patients carrying P. vivax malaria. Peripheral blood samples were collected from 155 P. vivax-infected individuals from five Brazilian malaria-endemic areas. The P. vivax CSP variants and the Duffy blood group genotypes were assessed using PCR/RFLP. In single infections, the VK210 variant was the commonest followed by the P. vivax-like variant. The typing of P. vivax indicated that the frequency of variants among the study areas was significantly different from one to another. This is the first detection of the VK247 and P. vivax-like variant in single infections in endemic areas of Brazil. Association of the CSP P. vivax variants with the heterozygous Duffy blood group system genotype was significant for VK210 single infection. These observations provide additional data on the Plasmodium-host interactions concerning the Duffy blood group and P. vivax capability of causing human malaria.