An algorithm to predict 3 ' intron splice sites in Plasmodium falciparum genomic sequences

A new algorithm, PfAGSS, for predicting 3' splice sites in Plasmodium falciparum genomic sequences is described. Application of this program to the published P. falciparum chromosome 2 and 3 data suggests that existing programs result in a high error rate in assigning 3' intron boundaries. (C) 2001 Elsevier Science B.V. All rights reserved.

Time of treatment influences the appearance of drug-resistant parasites in Plasmodium falciparum infections

A deterministic mathematical model which predicts the probability of developing a new drug-resistant parasite population within the human host is reported, The model incorporates the host's specific antibody response to PfEMP1, and also investigates the influence of chemotherapy on the probability of developing a viable drug-resistant parasite population within the host. Results indicate that early, treatment, and a high antibody threshold coupled with a long lag time between antibody stimulation and activity, are risk factors which increase the likelihood of developing a viable drug-resistant parasite population. High parasite mutation rates and fast PfEMP1 var gene switching are also identified as risk factors. The model output allows the relative importance of the various risk factors as well as the relationships between them to be established, thereby increasing the understanding of the conditions which favour the development of a new drug-resistant parasite population.

Alterations in Plasmodium falciparum genotypes during sequential infections suggest the presence of strain specific immunity

Many of the asexual stage Plasmodium falciparum proteins that are the targets of host protective responses are markedly polymorphic. The full repertoire of diversity is not defined for any antigen. Most studies have focused on the genes encoding merozoite surface proteins 1 and 2 (MSP1, MSP2). We explored the extent of diversity of some of the less studied merozoite surface antigens and analyzed the degree of complexity of malaria field isolates by deriving nucleotide sequences of several antigens. We have determined the genotype of apical membrane antigen 1 (AMA1) in a group of 30 field samples, collected over 29 months, from individuals living in an area of intense malaria transmission in Irian Jaya, identifying 14 different alleles. AMA1 genotyping was combined with previously determined MSP2 typing. AMA1 had the greatest power in distinguishing between isolates but methodological problems, especially when mixed infections are present, suggest it is not an ideal typing target. MSP1, MSP3, and glutamate-rich protein genotypes were also determined from a smaller group of samples, and all results were combined to derive an extended antigenic haplotype. Within this subset of 10 patients, nine different genotypes could be discerned; however, five patients were all infected with the same strain. This strain was present in individuals from two separate villages and was still present 12 months later. This strain was predominant at the first time point but had disappeared at the fourth time point. This significant change in malaria genotypes could be due to strain-specific immunity developing in this population.

"In vitro" activity of gentian violet against asexual Plasmodium falciparum parasites

In order to investigate whether gentian violet exhibited "in vitro" inhibitory activity against Plasmodium falciparum, the Authors have carried out 20 sensitivity tests according to the microtechnique described by RIECK MANN et al.5. Results have shown inhibition of schizonts'maturation at the following concentration: 1/1000; 1/1500; 1/2000; 1/2500; 1/3000 and 1/4000, thus demonstrating inhibitory activity of the tested dye against asexual blood parasites. The present data suggest gentain violet may be possibly used in the prophylaxis of transfusion-acquired malaria.

Plasmodium falciparum drug resistance in Angola

Facing chloroquine drug resistance, Angola promptly adopted artemisinin-based combination therapy as the first-line to treat malaria. Currently, the country aims to consolidate malaria control, while preparing for the elimination of the disease, along with others African countries in the region. However, the remarkable capacity of Plasmodium to develop drug resistance represents an alarming threat for those achievements. Herein, the available, but relatively scarce and dispersed, information on malaria drug resistance in Angola, is reviewed and discussed. The review aims to inform but also to encourage future research studies that monitor and update the information on anti-malarial drug efficacy and prevalence of molecular markers of drug resistance, key fields in the context and objectives of elimination.

Drug susceptibility of Plasmodium falciparum in the western Amazon region, state of Acre, Brazil

Field studies in the western Amazon region (state of Acre, Brazil) indicate that the 4-aminoquinolines, as well as the combined regimen with sulfadoxine-pyrimethamine, can no longer be recomended for the treatment and prophylaxis of P. falciparum infections in this region. Quinine remains an effective drug when used correctly. However, compliance problems arise due to the often occurring side-effects during a ten day regimen. Prospects of overcoming these constraints by combining a short course of quinine with other drugs are limited, because of the lack of suitable partner compounds. For this reason quinine/clindamycin appears to be a more practical therapy of P. falciparum malaria. In vitro data from this study suggest that mefloquine is another effective alternative for the treatment of falciparum malaria in this Amazon region.

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 (4x3), (EENVEHDA)2 (8x2) and (DDEHVEEPTVA)2(11x2) 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 8x2 peptides, and with 4x3 and 8x2 peptides in V. Paraíso, with a decrease in 11x2. MIFA titers appeared to be correlated mainly to the peptide 4x3 and it was the immunodominant in the three localities.

In vivo and in vitro Plasmodium falciparum Resistance to Chloroquine, Amodiaquine and Quinine in the Brazilian Amazon

In order to study the chemoresistance of Plasmodium falciparum to commonly used antimalarial drugs in Brazil the authors have studied ten patients with falciparum malaria, acquired in the Brazilian Amazon region. Patients were submitted to in vivo study of drug sensitivity, after chemotherapy with either 4-aminoquinolines (chloroquine or amodiaquine) or quinine. Adequate drug absorption was confirmed by standard urine excretion tests for antimalarials. Eight patients could be followed up to 28 days. Among these in vivo resistance (R I and R II responses) was seen in all patients who received 4-amino-quinolines. One patient treated with quinine exhibited a R III response. Peripheral blood samples of the same patients were submitted to in vitro microtests for sensitivity to antimalarials. Out of nine successful tests, resistance to chloroquine and amodiaquine was found in 100% and resistance to quinine in 11.11% of isolates. Probit analysis of log dose-response was used to determine effective concentrations EC50, EC90 and EC99 to the studied drugs. Good correlation between in vivo and in vitro results was seen in six patients. The results emphasize high levels of P. falciparum resistance to 4- aminoquinolines and suggest an increase in resistance to quinine in the Brazilian Amazon region, reinforcing the need for continuous monitoring of drug sensitivity to adequate chemotherapy according to the most efficacious drug regimens

STANDARDIZATION OF PROCEDURES OF Plasmodium falciparum ANTIGEN PREPARATION FOR SEROLOGIC TESTS

The objective of the present study is to standardize the technical variables for preparation and storage of Plasmodium falciparum and of antigen components extracted with the amphoteric detergent Zwittergent. P. falciparum obtained from in vitro culture was stored at different temperatures and for different periods of time. For each variable, antigen components of the parasite were extracted in the presence or absence of protease inhibitors and submitted or not to later dialysis. Products were stored for 15, 30 and 60 days at different temperatures and immunological activity of each extract was determined by SDS-PAGE and ELISA using positive or negative standard sera for the presence of IgG directed to blood stage antigens of P. falciparum. Antigen extracts obtained from parasites stored at -20oC up to 10 days or at -70oC for 2 months presented the best results, showing well-defined bands on SDS-PAGE and Western blots and presenting absorbance values in ELISA that permitted safe differentiation between positive and negative sera.

In vitro evaluation of quinidine sensitivity in brazilian Plasmodium falciparum isolates: comparative analysis to quinine and chloroquine

Falciparum malaria represents a serious and an increasing world public health problem due to the acquired parasite's resistance to the most available drugs. In some endemic areas, quinidine, a diastereoisomer of the antimalarial quinine, has been employed for replacing the latter. In order to evaluate the use of quinidine as an alternative to the increasing loss of quinine effectiveness in Brazilian P. falciparum strains, as has been observed in the Amazon area, we have assayed quinidine, quinine and chloroquine. The in vitro microtechnique was employed. All isolates showed to be highly resistant to chloroquine. Resistance to quinine was not noted although high MIC (minimal inhibitory concentration) values have been observed. These data corroborate the decreasing sensitivity to quinine in strains from Brazil. Quinidine showed IC50 from 0.053 to 4.577 mumol/L of blood while IC50 from 0.053 to 8.132 mumol/L of blood was estimated for quinine. Moreover, clearance of the parasitemia was observed in concentrations lower than that used for quinidine in antiarrhythmic therapy, confirming our previous data. The results were similar to African isolate.

Sharing of antigens between Plasmodium falciparum and Anopheles albimanus

The presence of common antigens between Plasmodium falciparum and Anopheles albimanus was demonstrated. Different groups of rabbits were immunized with: crude extract from female An. albimanus (EAaF), red blood cells infected with Plasmodium falciparum (EPfs), and the SPf66 synthetic malaria vaccine. The rabbit's polyclonal antibodies were evaluated by ELISA, Multiple Antigen Blot Assay (MABA), and immunoblotting. All extracts were immunogenic in rabbits according to these three techniques, when they were evaluated against the homologous antigens. Ten molecules were identified in female mosquitoes and also in P. falciparum antigens by the autologous sera. The electrophoretic pattern by SDS-PAGE was different for the three antigens evaluated. Cross-reactions between An. albimanus and P. falciparum were found by ELISA, MABA, and immunoblotting. Anti-P. falciparum and anti-SPf66 antibodies recognized ten and five components in the EAaF crude extract, respectively. Likewise, immune sera against female An. albimanus identified four molecules in the P. falciparum extract antigen. As far as we know, this is the first work that demonstrates shared antigens between anophelines and malaria parasites. This finding could be useful for diagnosis, vaccines, and the study of physiology of the immune response to malaria.

MOLECULAR SURVEILLANCE OF Plasmodium vivax AND Plasmodium falciparum DHFR MUTATIONS IN ISOLATES FROM SOUTHERN IRAN

In Iran, both Plasmodium vivax and P. falciparum malaria have been detected, but P. vivax is the predominant species. Point mutations in dihydrofolate reductase (dhfr) gene in both Plasmodia are the major mechanisms of pyrimethamine resistance. From April 2007 to June 2009, a total of 134 blood samples in two endemic areas of southern Iran were collected from patients infected with P. vivax and P. falciparum. The isolates were analyzed for P. vivax dihydrofolate reductase (pvdhfr) and P. falciparum dihydrofolate reductase (pfdhfr) point mutations using various PCR-based methods. The majority of the isolates (72.9%) had wild type amino acids at five codons of pvdhfr. Amongst mutant isolates, the most common pvdhfr alleles were double mutant in 58 and 117 amino acids (58R-117N). Triple mutation in 57, 58, and 117 amino acids (57L/58R/117N) was identified for the first time in the pvdhfr gene of Iranian P. vivax isolates. All the P. falciparumsamples analyzed (n = 16) possessed a double mutant pfdhfrallele (59R/108N) and retained a wild-type mutation at position 51. This may be attributed to the fact that the falciparum malaria patients were treated using sulfadoxine-pyrimethamine (SP) in Iran. The presence of mutant haplotypes in P. vivax is worrying, but has not yet reached an alarming threshold regarding drugs such as SP. The results of this study reinforce the importance of performing a molecular surveillance by means of a continuous chemoresistance assessment.

Genetic diversity and differentiation in natural Plasmodium falciparum populations inferred by molecular typing of the merozoite surface proteins 1 and 2

Genetic diversity and differentiation, inferred by typing the polymorphic genes coding for the merozoite surface proteins 1 (Msp-1) and 2 (Msp-2), were compared for 345 isolates belonging to seven Plasmodium falciparum populations from three continents. Both loci yielded similar estimates of genetic diversity for each population, but rather different patterns of between-population differentiation, suggesting that natural selection on these loci, rather than the transmission dynamics of P. falciparum, determines the variation in allele frequencies among populations.

In vitro evaluation of verapamil and other modulating agents in Brazilian chloroquine-resistant Plasmodium falciparum isolates

Verapamil, was assayed to record its modulating effect upon Brazilian Plasmodium falciparum isolates resistant to chloroquine. Other cardiovascular drugs known to be modulating agents in resistant malaria and/or multidrug-resistant neoplasias, including nifedipine, nitrendipine, diltiazem and propranolol, were also evaluated. Concentrations similar to those for cardiovascular therapy were used in the in vitro microtechnique for antimalarial drug susceptibility. Intrinsic antiplasmodial activity was observed from the lowest concentrations without a significant modulating action. Other reported modulating agents, such as the antipsychotic drug trifluoperazine and the antidepressants desipramine and imipramine, demonstrated similar responses under the same experimental conditions. Results suggest a much higher susceptibility of Brazilian strains, as well as an indifferent behaviour in relation to modulating agents.