The genetic diversity of Plasmodium malariae and Plasmodium brasilianum from human, simian and mosquito hosts in Brazil.

Plasmodium malariae is a protozoan parasite that causes malaria in humans and is genetically indistinguishable from Plasmodium brasilianum, a parasite infecting New World monkeys in Central and South America. P. malariae has a wide and patchy global distribution in tropical and subtropical regions, being found in South America, Asia, and Africa. However, little is known regarding the genetics of these parasites and the similarity between them could be because until now there are only a very few genomic sequences available from simian Plasmodium species. This study presents the first molecular epidemiological data for P. malariae and P. brasilianum from Brazil obtained from different hosts and uses them to explore the genetic diversity in relation to geographical origin and hosts. By using microsatellite genotyping, we discovered that of the 14 human samples obtained from areas of the Atlantic forest, 5 different multilocus genotypes were recorded, while in a sample from an infected mosquito from the same region a different haplotype was found. We also analyzed the longitudinal change of circulating plasmodial genetic profile in two untreated non-symptomatic patients during a 12-months interval. The circulating genotypes in the two samples from the same patient presented nearly identical multilocus haplotypes (differing by a single locus). The more frequent haplotype persisted for almost 3 years in the human population. The allele Pm09-299 described previously as a genetic marker for South American P. malariae was not found in our samples. Of the 3 non-human primate samples from the Amazon Region, 3 different multilocus genotypes were recorded indicating a greater diversity among isolates of P. brasilianum compared to P. malariae and thus, P. malariae might in fact derive from P. brasilianum as has been proposed in recent studies. Taken together, our data show that based on the microsatellite data there is a relatively restricted polymorphism of P. malariae parasites as opposed to other geographic locations.

Species-specific identification of Leishmania in naturally infected sand flies captured in Mato Grosso do Sul State, Brazil.

An increase in cutaneous and visceral leishmaniasis cases has been reported in recent years in the state of Mato Grosso do Sul, Brazil, and little is known to date about their etiological agents. An investigation into natural Leishmania infection of sand flies captured in this state between December 2003 and August 2004 was carried out. Mini-exon sequences were used as targets to identify Leishmania, and an RFLP technique was employed for those identified as belonging to the Viannia subgenus. Calculation of the minimal infection rate (MR) revealed that 1.6% of sand flies captured in the forest, peridomicile and intradomicile were positive. Six species were found to be infected by Leishmania (V.) braziliensis. Interestingly, two of the six species, Lutzomyia longipalpis and Nyssomyia whitmani, were captured in anthropic environments. The findings of this study constitute a useful tool for planning control measures against this disease in the State of Mato Grosso do Sul.

Population dynamics of genetically diverse Plasmodium falciparum lineages: community-based prospective study in rural Amazonia.

Temporal changes in the prevalence of antigenic variants in Plasmodium falciparum populations have been interpreted as evidence of immune-mediated frequency-dependent selection, but evolutively neutral processes may generate similar patterns of serotype replacement. Over 4 years, we investigated the population dynamics of P. falciparum polymorphisms at the community level by using 11 putatively neutral microsatellite markers. Plasmodium falciparum populations were less diverse than sympatric P. vivax isolates, with less multiple-clone infections, lower number of alleles per locus and lower virtual heterozygosity, but both species showed significant multilocus linkage disequilibrium. Evolutively neutral P. falciparum polymorphisms showed a high turnover rate, with few lineages persisting for several months in the population. Similar results had previously been obtained, in the same community, for sympatric P. vivax isolates. In contrast, the prevalence of the 2 dimorphic types of a major antigen, MSP-2, remained remarkably stable throughout the study period. We suggest that the relatively fast turnover of parasite lineages represents the typical population dynamics of neutral polymorphisms in small populations, with clear implications for the detection of frequency-dependent selection of polymorphisms.

Detection and identification of Leishmania species in field-captured phlebotomine sandflies based on mini-exon gene PCR.

Leishmaniasis is one of the most diverse and complex of all vector-borne diseases. Because it involves several overlapping species and sandfly vectors, the disease has a complex ecology and epidemiology. Adequate therapy and follow-up depend on parasitological diagnosis, but classical methods present several constraints when identifying species. We describe a polymerase chain reaction (PCR) which uses primers designed from mini-exon repetitive sequences that are specific for subgenus LeishmaniaViannia (PV), as well as sequences with specificity for genus (PG) that can distinguish between Leishmania species from other insect flagellates with minor differences in PCR products. For standardization, these PCR were tested in experimentally infected sandflies, and Leishmania infection in these insects was successfully confirmed. This methodology identified a 3.9% infection rate in field-captured phlebotomine sandflies from an endemic region in Brazil. Natural infection by Leishmania species was identified in three samples of Lutzomyia longipalpis, of which two were Leishmania (L.) chagasi and one Leishmania (L.) amazonensis. Irrespective of specific epidemiological conclusions, the method used in this study was able to identify Leishmania infections both in experimentally infected and field-captured phlebotomine sandflies, and could be a useful tool in epidemiological studies and strategic planning for the control of human leishmaniasis.

Amazonian malaria vector anopheline relationships interpreted from ITS2 rDNA sequences.

Species identification of anopheline mosquitoes (Diptera: Culicidae) can be problematic because many of them belong to complexes of morphologically similar species, often with contrasted ecology, behaviour and vectorial importance. The application of DNA-based diagnostics has proved to be useful for distinguishing between such species. We determined ribosomal DNA sequences of the second internal transcribed spacer (ITS2) from samples of 16 species of Anopheles captured in the Amazon Basin, Brazil. Length of the ITS2 varied from 323 to 410 base pairs, with GC content ranging from 50.7% to 66.5% and sequence identity from 25% to 99% between species. Maximum-likelihood paup analysis separated two distinct groups of species conforming with the recognized subgenera Anopheles (represented by eiseni, mattogrossensis, mediopunctatus and peryassui) and Nyssorhynchus (represented by 12 spp.). For the latter group, the neighbour-joining tree generated from rDNA sequence ITS2 relationships is compatible with the morphological taxonomic key established for these Amazonian species: albitarsis, aquasalis, benarrochi, braziliensis, darlingi, deaneorum, dunhami, evansae, nuneztovari, oswaldoi, rangeli and triannulatus. These ITS2 sequence data proved to be a useful tool for species identification and, potentially, to solve taxonomic problems.

Sequence analysis of the second internal transcribed spacer of ribosomal DNA in Anopheles oswaldoi (Diptera: Culicidae).

Sequence divergence in the second internal transcribed spacer (ITS2) of ribosomal DNA was examined for female specimens of Anopheles oswaldoi Peryassu from 7 localities in South America. The lengths of ITS2 for all mosquitoes ranged from 348 to 356 nucleotides. After alignment of these sequences, similarity ranged from 87 to 100%. Divergence was within the range of inter-specific differences for members of anopheline species complexes. Therefore, specimens were placed into 4 groups that may correspond to at least 4 cryptic species. One is probably related to An. oswaldoi sensu stricto and another to Anopheles konderi Galvão & Damasceno. The other 2 groups may correspond to species for which morphological identification remains to be clarified. These data provide evidence that An. oswaldoi comprise a complex of cryptic species and that DNA identification may help to resolve the taxonomic questions related to this group.

Analysis of ITS2 DNA sequences from Brazilian Anopheles darlingi (Diptera: Culicidae).

Specimens of Anopheles darlingi Root, the major vector of malaria in Brazil, were collected from several states in Brazil: Sao Paulo (Dourado), Bahia (Itabela), Rondônia (Porto Velho), Roraima (Boa Vista), and Acre (Plácido de Castro). Sequence divergence in the 2nd internal transcribed spacer (ITS2) was examined. The ITS2 sequences of mosquitoes captured in the Amazon region (Porto Velho, Boa Vista and Plácido de Castro) and in the northeast of Brazil (Itabela) were almost identical; however, a 4-5% sequence divergence was observed in the ITS2 of mosquitoes captured in the southeast (Dourado). Further analysis is needed to determine if these differences indicate that Dourado population may be a separate species.

Seasonal variation of anti-Plasmodium falciparum antibodies directed against a repetitive peptide of gametocyte antigen pfs2400 in inhabitants in the State of Amapá, Brazil.

Antibodies to the Pfs2400 gametocyte antigen have been shown to inhibit the development of Plasmodium falciparum in anophelines and therefore this antigen is a candidate for a transmission-blocking vaccine. To test seasonal variation of these antibodies under field conditions, sera from 72 individuals were collected twice, first during the long-rains season with low malaria transmission and then, 6 months later, during the short-rains season, when transmission is high. This study was conducted in several localities in the State of Amapá, Brazil. All but three individuals had a positive indirect fluorescent antibody test (IFAT) with asexual forms of P. falciparum. Most of them did not report malaria attacks during the period between the first and second sampling. Their sera were tested by IFAT with P. falciparum gametocytes. The overall positivity of this test did not vary between seasons, and was 47.2 (34/72) and 48.6% (35/72), respectively. The sera were also tested by ELISA with the Pfs2400 repeat peptide. The positivity rate dropped from 29.2 (21/72) to 15.3% (11/72) and the mean absorbancies from 0.623 to 0.354, when we compared the results of the long-rains and short-rains seasons. Fifteen out of the 21 ELISA positive sera turned negative, with no change of geometric mean of titres (GMT) of asexual IFAT, while five negatives became ELISA positive on second sampling, with increase of GMT. Soon after the second sampling a malaria outbreak was reported in one of the localities. These results point toward a relatively short persistence of anti-Pfs2400 repeat peptide antibodies, under natural field conditions. A gametocyte antigen booster before a high transmission period might contribute towards lowering malaria incidence by eliciting a partially effective antibody response.

Seasonal variation of anti-RESA/Pf155 Plasmodium falciparum antibodies in three localities from the state of Amapá, Brazil.

Anti-RESA/Pf155 antibodies were assayed in sera of individuals from three localities (Laranjal do Jari, Vila Padaria and Vila Paraíso) in the State of Amapá, Brazil, during the long-rains and short-rains seasons. All of these had negative blood smears for malaria. Most of the sera collected were positive in Indirect Fluorescent Antibody (IFA) with P. falciparum parasites, with no seasonal variation. A high percentage of these sera (62% to 100%) was RESA positive by Modified Indirect Fluorescent Antibody (MIFA), with a significant (p < 0.05) increase of geometric mean titers during the short-rains season, when the transmission of the disease is highest. ELISA with three repetitive RESA peptides (EENV)3 (4 x 3), (EENVEHDA)2 (8 x 2) and (DDEHVEEPTVA)2 (11 x 2) did not reveal statistically significant seasonal variations, although a small enhancement of positivity was observed in V. Padaria (15.3 to 38.8%) in the short-rains season with the 8 x 2 peptides, and with 4 x 3 and 8 x 2 peptides in V. Paraíso, with a decrease in 11 x 2. MIFA titers appeared to be correlated mainly to the peptide 4 x 3 and it was the immunodominant in the three localities.