Effect of synthesized inhibitors on babesipain-1, a new cysteine protease from the bovine piroplasm Babesia bigemina.

Papain-like cysteine proteases (CP) have been shown to have essential roles in parasitic protozoa and are under study as promising drug targets. One gene was identified by sequence similarity search to be homologous to the CP family in the ongoing Babesia bigemina genome sequencing project database. The newly identified CP gene, called babesipain-1, was cloned and expressed as a fusion protein, and the effect of different inhibitors on proteolytic activity was tested. A series of new artemisinin-vinyl sulfone hybrid molecules were tested as inhibitors being effective on the range of 0.3-30 microm, depending on the core-containing molecule.

Differential expression of cytokine genes among sickle-cell-trait (HbAS) and normal (HbAA) children infected with Plasmodium falciparum.

The human immune response to Plasmodium falciparum infection involves the release of cytokines that may contribute to the control of the parasites' replication. These cytokines are also involved in the pathogenesis of the malaria caused by the infection, leading to the appearance of symptoms of varying severity. In a cross-sectional study, the expression of the genes that code for pro-inflammatory cytokines (tumour necrosis factor, interferon-gamma, interleukin-6 and interleukin-12) and anti-inflammatory cytokines (interleukin-10 and interleukin-4) among 80 children infected with P. falciparum (from a malaria-endemic area of Sudan) and five healthy controls (from a non-endemic area) was explored. The infected children were either non-sicklers, with severe malaria (18 children), mild malaria (30) or no symptoms of malaria (18), or asymptomatic sicklers (14). Interleukin-12 was found to be weakly expressed by all the groups of children. In general, compared with the other groups, the asymptomatic non-sicklers had lower expression of all the cytokines studied. The asymptomatic sicklers had significantly lower expression of tumour necrosis factor than the non-sicklers with severe malaria, but these two groups showed similar expression of interferon-gamma, interleukin-4 and interleukin-6. Gene expression of the regulatory cytokine, interleukin-10, by the asymptomatic sicklers was significantly lower than that by the non-sicklers with severe malaria but higher than that recorded in the non-sicklers with mild malaria. Their regulation of cytokine release appears to protect sicklers from clinical malaria.

Present habitat suitability for Anopheles atroparvus (Diptera, Culicidae) and its coincidence with former malaria areas in mainland Portugal.

Malaria was a major health problem in the first half of the 20th Century in mainland Portugal. Nowadays, although the disease is no longer endemic, there is still the risk of future endemic infections due to the continuous occurrence of imported cases and the possibility of transmission in the country by Anopheles atroparvus Van Thiel, 1927. Since vector abundance constitute one of the foremost factors in malaria transmission, we have created several habitat suitability models to describe this vector species' current distribution. Three different correlative models; namely (i) a multilayer perceptron artificial neural network (MLP-ANN); (ii) binary logistic regression (BLR); and (iii) Mahalanobis distance were used to combine the species records with a set of five environmental predictors. Kappa coefficient values from k-fold cross-validation records showed that binary logistic regression produced the best predictions, while the other two models also produced acceptable results. Therefore, in order to reduce uncertainty, the three suitability models were combined. The resulting model identified high suitability for An. atroparvus in the majority of the country with exception of the northern and central coastal areas. Malaria distribution during the last endemic period in the country was also compared with the combined suitability model, and a high degree of spatial agreement was obtained (kappa = 0.62). It was concluded that habitat suitability for malaria vectors can constitute valuable information on the assessment of several spatial attributes of the disease. In addition, the results suggest that the spatial distribution of An. atroparvus in the country remains very similar to the one known about seven decades ago.

The relevance of molecular markers in the analysis of malaria parasite populations.

Malaria is one of the main human public health problems in the tropical world and is possibly becoming an emerging disease too in regions where it has been controlled. It has been an excellent model in the area of molecular studies, with scientific validation of techniques, application of data mainly in studies of parasite diversity and information on a number of different aspects associated with infection and disease. The transfer of the gathered knowledge and experience in malaria to other infections is of great use and we briefly review a number of molecular markers, methodologies and techniques mostly used for Plasmodium detection, as well as identification or characterization of parasite populations. Selection of appropriate techniques depends on the questions raised and the studies' objectives--the antigen-coding genes, microsatellite loci and drug-resistance associated markers being the three most analysed classes of markers. The need of validation and standardization of laboratory protocols is addressed and discussed as it may determine the comparison of data between different studies and laboratories, with relevance in field-collected samples or studies.

A primer-introduced restriction analysis-polymerase chain reaction method to detect knockdown resistance mutations in Anopheles gambiae.

In the major malaria vector Anopheles gambiae Giles, two point mutations at the voltage-gated sodium channel have been associated with knockdown resistance (kdr) to DDT and pyrethroid insecticides. Simple allele-specific polymerase chain reaction (PCR) assays to detect these single-nucleotide polymorphisms are prone to lack of specificity and therefore alternative techniques have been proposed. However, these may not be easily implemented in many laboratories from malaria endemic regions. Here, we describe a primer-introduced restriction analysis (PIRA)-PCR method to detect kdr mutations in An. gambiae. This method unambiguously identified all six genotypes for the kdr locus in a sample of 113 field-collected mosquitoes for which kdr genotypes had been confirmed by DNA sequencing. Co-occurrence of both kdr alleles was found in sites from Equatorial Guinea and Gabon and the L1014F mutation was detected in M-form individuals from Angola. The PIRA-PCR proved to be a reliable, robust, and simpler alternative for the detection of kdr mutations in this malaria vector.

Studies on antimalarial drug susceptibility in Colombia, in relation to Pfmdr1 and Pfcrt.

In Colombia, Plasmodium resistance to antimalarials such as chloroquine and antifolates is a serious problem. As a result, the national Colombian health authorities are monitoring the efficacy of alternative drugs and schemes. The study of genetic polymorphisms related with drug resistance is required in the region. In vitro responses to chloroquine, quinine, mefloquine, amodiaquine, desethylamodiaquine, artesunate and dihydroartesunate were carried out by HRP ELISA. SNP analysis in Pfcrt and Pfmdr1 genes was performed by PCR-RFLP in 77 samples from the North West region of Colombia. In vitro resistance to chloroquine was high (74%), followed by mefloquine (30%) and desethylamodiaquine (30%). A positive correlation between the IC(50) of paired drugs was also detected. The allele Pfmdr1 N86 (wild) was present in 100% of the samples and 1246Y (mutant) in 92%. However, their presence did not correlate with in vitro drug resistance. Presence of the mutations K76T and N75E in Pfcrt was confirmed in all samples. Analysis of 4 codons (72, 74, 75 and 76) in pfcrt confirmed the presence of the haplotypes CMET in 91% and SMET in 9% of the samples.

Knockdown resistance mutations (kdr) and insecticide susceptibility to DDT and pyrethroids in Anopheles gambiae from Equatorial Guinea.

OBJECTIVES: To determine the frequency of knockdown resistance (kdr) mutations in the malaria vector Anopheles gambiae s.s. from continental Equatorial Guinea; and to relate kdr genotypes with susceptibility to DDT and pyrethroid insecticides in this vector. METHODS: Female mosquitoes were collected in two villages, Miyobo and Ngonamanga, of mainland Equatorial Guinea. Insecticide susceptibility tests were performed following WHO procedures. Anopheles gambiae complex specimens were identified to species and molecular form by PCR. Genotyping of the kdr locus was performed by allele-specific PCR and direct sequencing in a subset of samples. RESULTS: Both M and S molecular forms of A. gambiae were found in Ngonamanga whereas only the S-form was identified in Miyobo. The two kdr mutations were detected in S-form samples of both villages, with a higher frequency of the kdr-e (Leu-1014-Ser) allele (Miyobo: 16%; Ngonamanga: 40%). The kdr-w (Leu-1014-Phe) mutation was also detected in 3% of the M-form. All individuals tested for pyrethroids were susceptible. A mortality rate of 86% was obtained for DDT. An overall kdr allele frequency (i.e. kdr-e + kdr-w) of 22% was detected in DDT resistant individuals, whereas susceptible individuals had a kdr frequency of 6%. CONCLUSION: The co-occurrence of both kdr mutations and reduced susceptibility to DDT found in A. gambiae highlights the importance of implementing efficient surveillance of insecticide resistance in Equatorial Guinea.

Co-occurrence of East and West African kdr mutations suggests high levels of resistance to pyrethroid insecticides in Anopheles gambiae from Libreville, Gabon.

Point mutations in the voltage-gated sodium channel gene involved in knockdown resistance to DDT and pyrethroid insecticides have been described in several insect species. In the malaria vector Anopheles gambiae Giles sensu stricto (Diptera: Culicidae) two mutations have been identified. The first, consisting of a leucine-phenylalanine substitution at amino acid position 1014, is widespread in West Africa. The second, a leucine-serine substitution at the same position, has to date only been detected in western Kenya. Analysis of the kdr polymorphism in a sample of 106 An. gambiae s.s. of the rDNA S-form/Type I collected in Libreville (Gabon) surprisingly revealed the presence of both East and West African kdr mutations with frequencies of 63% and 37%, respectively. No wild-type alleles were detected and there was an excess of heterozygous genotypes (P = 0.04). In addition, an inconsistency was found during the kdr genotyping procedures by polymerase chain reaction, which could have lead to an underestimation of resistance alleles. The implications of these findings are discussed.

Effect of antibodies on the expression of Plasmodium falciparum circumsporozoite protein gene.

Antibodies are known to play an important role in the control of malaria infection. However, they can modulate parasite development enhancing infection. The effect of anti-Plasmodium antibodies on the expression of circumsporozoite protein gene (csp) was investigated. Plasmodium falciparum 3D7 in vitro cultures were submitted to: i) anti- circumsporozoite protein monoclonal antibody (anti-CSP-mAb) [1microg/ml, 0.1microg/ml, 0.01microg/ml and 0.001microg/ml] and ii) purified IgG Fab fragment from a pool of malaria patients [1mg/ml and 1microg/ml]; and compared to control cultures. After 24h the number of ring infected erythrocytes was determined in order to calculate invasion efficacy. At 48h culture supernatant was collected, and the amount of circumsporozoite protein determined by ELISA, parasitaemia was calculated and cells were processed for RNA preparation. Expression of csp gene was quantified using Real time RT-PCR. There was an increase in parasite growth when treated with lower anti-CSP-mAb concentration, which was associated with lower csp expression, while 1mug/ml anti-CSP-mAb treatment presented a growth inhibitory effect accompanied by high csp expression.

Effect of chloroquine on the expression of genes involved in the mosquito immune response to Plasmodium infection.

Chloroquine has been described to increase Plasmodium infectivity to the mosquito vector and is known to affect the vertebrate host immune response including during malarial infection. Although knowledge of the mosquito immune response has recently improved, nothing is known about the impact of chloroquine on mosquito immunity. In order to characterize the influence of chloroquine on the mosquito immune system, we have analyzed the effect of chloroquine on Anopheles gambiae (i) serine proteases and (ii) antimicrobial peptide gene expression, in uninfected and Plasmodium berghei infected mosquitoes, using real-time PCR. We have demonstrated for the first time that mosquitoes fed on chloroquine-treated mice showed a significant down regulation of some immune-related genes. This effect was independent of midgut bacterial burden. These results suggest that chloroquine might act on the Anopheles serine proteases cascade, interfering with signal transduction pathways and at a transcriptional activation level.

Mating does not affect the biting behaviour of Anopheles gambiae from the islands of São Tomé and Príncipe, West Africa.

To determine if mating or gonotrophic age influenced the biting behaviour of Anopheles gambiae s.s., a series of all-night landing captures was performed on the islands of São Tomé and Príncipe in the Gulf of Guinea. On São Tomé 49% and on Príncipe 56% of the newly emerged An. gambiae taking their first bloodmeal were virgins. On each island, with the exception of recently mated insects on Príncipe, all age-groups had similar biting cycles. The biting cycle on Príncipe resembled that observed on continental Africa, with a peak in the latter part of the night. Peak biting on São Tomé, however, occurred before midnight. Estimated daily survival rates were 0.77 and 0.29 for São Tomé and Príncipe, respectively. Mating does not affect the biting behaviour of An. gambiae on these islands.

An island within an island: genetic differentiation of Anopheles gambiae in São Tomé, West Africa, and its relevance to malaria vector control.

Islands are choice settings for experimental studies of vector control strategies based on transgenic insects. Before considering this approach, knowledge of the population structure of the vector is essential. Genetic variation at 12 microsatellite loci was therefore studied in samples of the malaria vector Anopheles gambiae s.s., collected from six localities of São Tomé island (West Africa). The objectives were (i) to assess the demographic stability and effective population size of A. gambiae from these sites, (ii) to determine population differentiation and (iii) to relate the observed patterns of population structure with geographic, ecological and historical aspects of the vector on the island. Significant population differentiation, revealed by FST and RST statistics, was found between the southernmost site, Porto Alegre, and northern localities. The observed patterns of population substructure are probably a result of restrictions to gene flow in the less inhabited, more densely forested and mountainous south. In all localities surveyed, A. gambiae appeared to be experiencing a demographic expansion, consistent with a relatively recent (ca. 500 years) founder effect. The results are discussed with respect to current and future prospects of malaria vector control.

[New concepts in hyperactive malarial splenomegaly]. / Novos conceitos na esplenomegalia malárica hiperreactiva.

Hyperreactive malarial splenomegaly is thought to represent an immunological dysfunction due to recurrent episodes of malaria. The authors present a case of hyperreactive malarial splenomegaly in a patient from São Tomé e Príncipe and discuss aspects of its differential diagnosis and treatment. A revision is made of recent concepts related to its pathogenesis and relationship with lymphoproliferative disorders. Malarial DNA was found in the absence of parasite forms in the peripheral blood. This may indicate that latent infection plays a role in its pathogenesis.

Genetic structure of Anopheles gambiae (Diptera: Culicidae) in São Tomé and Príncipe (West Africa): implications for malaria control.

The impact of a vector eradication programme, conducted in the 1980s, on Anopheles gambiae populations from the islands of São Tomé and Príncipe, was evaluated by microsatellite DNA analysis. Significant genetic differentiation was observed within and between the two islands and between the islands and a population from Gabon, suggesting a degree of isolation between them. Large estimates of long-term N(e) suggested that the control programme did not affect the effective population size of the vector. Heterozygosity tests were also not consistent with a recent bottleneck.

Male size does not affect mating success (of Anopheles gambiae in São Tomé).

For malaria control, the utility of transgenic vector Anopheles mosquitoes (Diptera: Culicidae) refractory to Plasmodium transmission, will depend on their interbreeding with the wild vector population. In many species, larger males are more successful in obtaining mates. In São Tomé island, we determined that size did not affect mating success of male Anopheles gambiae Giles sensu stricto, the main malaria vector in tropical Africa. Also we showed that larval intraspecific competition is probably insignificant in this population of An. gambiae. Thus, the potential success of transgenic An. gambiae is unlikely to be affected by size selection under field conditions.

The swarming and mating behaviour of Anopheles gambiae s.s. (Diptera: Culicidae) from São Tomé Island.

The swarming and mating behaviours of the forest cytoform of Anopheles gambiae s.s. were investigated on 194 evenings and 14 mornings between April 1997 and November 1999 in a peri-urban area of the island of São Tomé, West Africa. Males swarmed 2-3 m above markers of horizontal contrast such as those formed between grass areas and footpaths, or bushes. Evening swarms started 2 min before sunset in sheltered sites and a minute or two later in exposed ones. It took approximately 5 mins from the arrival of the first male for the swarm to reach estimated maximum numbers. Mating pairs were first seen 7 min after the start of swarming. Maximum numbers of pairs in copula were observed 8 min later. Up to 270 pairings were seen in the 20 min period before darkness. Removal of males had no effect on the number of females arriving at the swarm. Males were attracted to sounds that approximated the female flight tone but not to filter paper samples of squashed virgin females swung through the swarm. A much-reduced amount of swarming and a single mating were recorded at dawn. The same locations for swarming were used at different times and at different heights by ants, Culicoides sp. and Culex quinquefasciatus.

Genotyping of Plasmodium falciparum infections by PCR: a comparative multicentre study.

Genetic diversity of malaria parasites represents a major issue in understanding several aspects of malaria infection and disease. Genotyping of Plasmodium falciparum infections with polymerase chain reaction (PCR)-based methods has therefore been introduced in epidemiological studies. Polymorphic regions of the msp1, msp2 and glurp genes are the most frequently used markers for genotyping, but methods may differ. A multicentre study was therefore conducted to evaluate the comparability of results from different laboratories when the same samples were analysed. Analyses of laboratory-cloned lines revealed high specificity but varying sensitivity. Detection of low-density clones was hampered in multiclonal infections. Analyses of isolates from Tanzania and Papua New Guinea revealed similar positivity rates with the same allelic types identified. The number of alleles detected per isolate, however, varied systematically between the laboratories especially at high parasite densities. When the analyses were repeated within the laboratories, high agreement was found in getting positive or negative results but with a random variation in the number of alleles detected. The msp2 locus appeared to be the most informative single marker for analyses of multiplicity of infection. Genotyping by PCR is a powerful tool for studies on genetic diversity of P. falciparum but this study has revealed limitations in comparing results on multiplicity of infection derived from different laboratories and emphasizes the need for highly standardized laboratory protocols.