The drug transporter ABCB1 c.3435C>T SNP influences artemether-lumefantrine treatment outcome.

Malaria treatment performance is potentially influenced by pharmacogenetic factors. This study reports an association study between the ABCB1 c.3435C>T, CYP3A4*1B (g.-392A>G), CYP3A5*3 (g.6986A>G) SNPs and artemether + lumefantrine treatment outcome in 103 uncomplicated malaria patients from Angola. No significant associations with the CYP3A4*1B and CYP3A5*3 were observed, while a significant predominance of the ABCB1 c.3435CC genotype was found among the recurrent infection-free patients (p < 0.01), suggesting a role for this transporter in AL inter-individual performance.

Evaluation of artemether-lumefantrine efficacy in the treatment of uncomplicated malaria and its association with pfmdr1, pfatpase6 and K13-propeller polymorphisms in Luanda, Angola.

BACKGROUND: Drug resistance in Plasmodium falciparum has posed an obstacle to effective treatment and challenges many malaria control programmes in endemic areas. In Angola, until 2003, chloroquine (CQ) was used as first-line therapy for uncomplicated malaria. It was replaced initially by amodiaquine and, in 2006, by artemisinin-based combination therapy (ACT) with artemether-lumefantrine (AL, Coartem(®)). Efficacy study of ACT, conducted in Angola between 2004 and 2005, showed a baseline efficacy of ≈99%. METHODS: 103 malaria patients were enrolled according to WHO proceedings. Patients were followed up with clinical and parasitological evaluations for 28 days, parasite density and identification was evaluated by microscopy, the pfmsp2 were genotyped by nested-PCR, to distinguish parasite recrudescence from new infections; the polymorphisms at codons 86 and 1246 of pfmdr1 gene, and 769 of pfatp6 gene were assessed by PCR-RFLP and sequencing for pfk13-propeller genotype. RESULTS: The cure rate was 91.3%. The obtained results showed that from 103 patients, 12.6% (n = 13) still had parasitaemia 1 day after the treatment was finished. On day 0, of the 94 evaluated samples, wild-type alleles were identified in 73.4% (n = 69) for pfmdr1 N86Y position and only one sample carried the mutant allele (Y) for pfmdr1 1246; 14% of these samples showed increased pfmdr1 copy number; 100% (n = 21) had wild-type allele of k13 gene in all the studied positions. DISCUSSION: These results showed changes in parasite profile susceptibility to AL in comparison to the baseline data from 2002 to 2004 and on the genotyping characteristics; the clinical outcome after treatment with AL did not link a particular genotype with treatment failure; observed changes do not provide sufficient evidence for a treatment policy change, but they suggest that a carefully monitoring is needed in this area.

Analysis of polymorphisms in Plasmodium falciparum genes related to drug resistance: a survey over four decades under different treatment policies in Brazil.

BACKGROUND: Anti-malarial resistance in Plasmodium falciparum remains an obstacle for malaria control. Resistance-associated genes were analysed in Brazilian samples over four decades to evaluate the impact of different treatment regimens on the parasite genetic profile. METHODS: Samples were collected on filter paper from patients infected in the Amazon region from 1984 to 2011. DNA was extracted with Chelex® 100 and monoinfection confirmed by PCR. SNPs in the pfcrt, pfmdr1, pfdhfr and pfdhps genes were assessed by PCR-RFLP. The pfmdr1 copy number was estimated using real time quantitative PCR with SYBR® Green. Parasite response was assessed ex vivo with seven concentrations of each anti-malarial. Patients were treated according to Brazilian guidelines: quinine plus tetracycline or mefloquine in period 1 and ACT in period 2. RESULTS: All 96 samples presented the pfcrt 76T mutant throughout the assessed periods. In addition, all isolates showed ex vivo chloroquine resistance. The pfmdr1 86Y was detected in 1.5% of samples in period 1, and in 25% in period 2. All samples presented the pfmdr1 1246Y. The analysis of pfmdr1 copy number showed amplification in 37.3% in period 1 and in 42% in period 2. Mutations in pfdhfr were shown as follows: 51I in all samples in period 1 and in 81.2% in period 2; 59R in 6.4% in period 2. The pfdhfr 108N and the pfdhps 437G were seen in all samples along time; the pfdhps 540E in 93.7% in period 1 and in 75% in period 2. CONCLUSIONS: The 76T mutation associated to chloroquine resistance is still present in the parasite population, although this anti-malarial was withdrawn from the chemotherapy of P. falciparum in Brazil in the mid-1980s. All isolates assayed ex vivo for chloroquine showed resistant phenotype and 76T. No association was observed between pfmdr1 mutations and resistance to quinine, mefloquine and artemisinin derivatives. Additionally, the pfdhfr 108N mutation was detected in all samples throughout the evaluated periods, demonstrating fixation of the mutant allele in the parasite population. Changes in Brazilian national guidelines for the malaria chemotherapy in the last 27 years yielded a discreet genetic impact in the parasite population.

Genetic diversity and signatures of selection of drug resistance in Plasmodium populations from both human and mosquito hosts in continental Equatorial Guinea.

BACKGROUND: In Plasmodium, the high level of genetic diversity and the interactions established by co-infecting parasite populations within the same host may be a source of selection on pathogen virulence and drug resistance. As different patterns have already been described in humans and mosquitoes, parasite diversity and population structure should be studied in both hosts to properly assess their effects on infection and transmission dynamics. This study aimed to characterize the circulating populations of Plasmodium spp and Plasmodium falciparum from a combined set of human blood and mosquito samples gathered in mainland Equatorial Guinea. Further, the origin and evolution of anti-malarial resistance in this area, where malaria remains a major public health problem were traced. METHODS: Plasmodium species infecting humans and mosquitoes were identified by nested-PCR of chelex-extracted DNA from dried blood spot samples and mosquitoes. Analysis of Pfmsp2 gene, anti-malarial-resistance associated genes, Pfdhps, Pfdhfr, Pfcrt and Pfmdr1, neutral microsatellites (STR) loci and Pfdhfr and Pfdhps flanking STR was undertaken to evaluate P. falciparum diversity. RESULTS: Prevalence of infection remains high in mainland Equatorial Guinea. No differences in parasite formula or significant genetic differentiation were seen in the parasite populations in both human and mosquito samples. Point mutations in all genes associated with anti-malarial resistance were highly prevalent. A high prevalence was observed for the Pfdhfr triple mutant in particular, associated with pyrimethamine resistance.Analysis of Pfdhps and Pfdhfr flanking STR revealed a decrease in the genetic diversity. This finding along with multiple independent introductions of Pfdhps mutant haplotypes suggest a soft selective sweep and an increased differentiation at Pfdhfr flanking microsatellites hints a model of positive directional selection for this gene. CONCLUSIONS: Chloroquine is no longer recommended for malaria treatment in Equatorial Guinea but sulphadoxine-pyrimethamine (SP) remains in use in combination with artesunate and is the only drug recommended in preventive chemotherapy in pregnancy. The high prevalence of point mutations in Pfdhfr and Pfdhps points to the danger of an eventual reduction in the efficacy of SP combined therapy in P. falciparum populations in Equatorial Guinea and to the essential continuous monitoring of these two genes.

Strengthening research capacity through the medical education partnership initiative: the Mozambique experience.

BACKGROUND: Since Mozambique's independence, the major emphasis of its higher educational institutions has been on didactic education. Because of fiscal and human resource constraints, basic and applied research activities have been relatively modest in scope, and priorities have often been set primarily by external collaborators. These factors have compromised the scope and the relevance of locally conducted research and have limited the impact of Mozambique's universities as major catalysts for national development. CASE DESCRIPTION: We developed a multi-institutional partnership to undertake a comprehensive analysis of the research environment at Mozambique's major public universities to identify factors that have served as barriers to the development of a robust research enterprise. Based on this analysis, we developed a multifaceted plan to reduce the impact of these barriers and to enhance research capacity within Mozambique. INTERVENTIONS: On the basis of our needs assessment, we have implemented a number of major initiatives within participating institutions to facilitate basic and applied research activities. These have included specialized training programmes, a reorganization of the research administration infrastructure, the development of multiple collaborative research projects that have emphasized local research priorities and a substantial investment in bioinformatics. We have established a research support centre that provides grant development and management services to Mozambique's public universities and have developed an independent Institutional Review Board for the review of research involving human research subjects. Multiple research projects involving both communicable and non-communicable diseases have been developed and substantial external research support has been obtained to undertake these projects. A sizable investment in biomedical informatics has enhanced both connectivity and access to digital reference material. Active engagement with relevant entities within the Government of Mozambique has aligned institutional development with national priorities. CONCLUSIONS: Although multiple challenges remain, over the past 3 years significant progress has been made towards establishing conditions within which a broad range of basic, translational and clinical and public health research can be undertaken. Ongoing development of this research enterprise will enhance capacity to address critical locally relevant research questions and will leverage resources to accelerate the development of Mozambique's national universities.

Novel potent metallocenes against liver stage malaria.

Novel conjugates of the antimalarial drug primaquine (compound 1) with ferrocene, named primacenes, have been synthesized and screened for their activities against blood stage and liver stage malaria in vitro and host-vector transmission in vivo. Both transmission-blocking and blood-schizontocidal activities of the parent drug were conserved only in primacenes bearing a basic aliphatic amine group. Liver stage activity did not require this structural feature, and all metallocenes tested were comparable to or better than primaquine in this regard. Remarkably, the replacement of primaquine's aliphatic chain by hexylferrocene, as in compound 7, led to a ~45-fold-higher level activity against liver stage parasitemia than that of primaquine.

Application of loop-mediated isothermal amplification for malaria diagnosis during a follow-up study in São Tomé.

BACKGROUND: A reliable and simple test for the detection of malaria parasite is crucial in providing effective treatment and therapeutic follow-up, especially in malaria elimination programmes. A comparison of four methods, including nested polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) were used for the malaria diagnosis and treatment follow-up in São Tomé and Príncipe, during a successful pre-elimination campaign. METHOD: During the period September to November 2009, blood samples from 128 children (five to 14 years old) with temperature ≥38°C (tympanic) in the District of Agua Grande were examined using four different methods, i.e., histidine-rich protein 2 (HRP-2) based rapid diagnostic tests (HRP-2-RDTs), optical microscopy, nested PCR, and LAMP. First-line treatment with artesunate-amodiaquine was given for uncomplicated malaria and intravenous quinine was given for complicated malaria. Children with persistent positivity for malaria by microscopy, or either by nested PCR, or by LAMP on day 7 were given second-line treatment with artemether-lumefantrine. Treatment follow-up was made weekly, for up to four weeks. RESULTS: On day 0, positive results for HRP-2-RDTs, microscopy, nested PCR, and LAMP, were 68(53%), 47(37%), 64(50%), and 65(51%), respectively. When nested PCR was used as a reference standard, only LAMP was comparable; both HRP-2-RDTs and microscopy had moderate sensitivity; HRP-2-RDTs had poor positive predictive value (PPV) and a moderate negative predictive value (NPV) for the treatment follow-up. Seventy-one children with uncomplicated malaria and eight children with complicated falciparum malaria were diagnosed based on at least one positive result from the four tests as well as clinical criteria. Twelve of the 79 children receiving first-line treatment had positive results by nested PCR on day 7 (nested PCR-corrected day 7 cure rate was 85%). After the second-line treatment, nested PCR/LAMP-corrected day 28 cure rate was 83% for these 12 children. CONCLUSIONS: HRP-2-RDTs have similar sensitivity as microscopy but less specificity. However, as compared to nested PCR, the poor sensitivity of HRP-2-RDTs indicates that low parasitaemia may not be detected after treatment, as well as the low specificity of HRP-2-RDTs indicates it cannot be applied for treatment follow-up. LAMP has similar sensitivity and specificity to nested PCR. With high PPV and NPV, LAMP is simpler and faster as compared to nested PCR with the advantage of detecting low parasitaemia becoming a potential point-of-care test for treatment follow-up.

A cross-sectional study of sub-clinical Plasmodium falciparum infection in HIV-1 infected and uninfected populations in Mozambique, South-Eastern Africa.

BACKGROUND: Plasmodium falciparum and HIV-1 infection cause substantial morbidity and mortality in sub-Saharan Africa. Increasing evidence suggests these two pathogens interact negatively when infecting the same individual. METHODS: A cross-sectional study among HIV-1 infected and uninfected populations was recruited in Mocuba and Maputo, Mozambique to determine the prevalence of sub-clinical malarial parasitaemia using light microscopy and a nested PCR assay. RESULTS: The prevalence of sub-clinical P. falciparum parasitaemia was low in Maputo, whether determined by microscopy (0.4%) or PCR (1.9%), but substantially higher in Mocuba (7.6 and 14.7%, respectively). Nested PCR detected nearly 70% more cases of sub-clinical parasitaemia than microscopy, but differences occur by locality. HIV-1 infected persons were more likely to be sub-clinically parasitaemic than HIV-1 uninfected individuals recruited from the same geographic areas. Trimethoprim-sulphamethoxazole use did not substantially reduce sub-clinical parasitaemia. CONCLUSIONS: Dried blood spots are a convenient and sensitive technique for detecting sub-clinical infection with P. falciparum by nested PCR. Prevalence of P. falciparum is substantially lower in Maputo where malaria control programmes have been more active than in the rural town of Mocuba. In Mocuba, among those presenting for HIV-1 counseling and testing, the prevalence of P. falciparum is substantially higher in those who test positive for HIV-1 than those without HIV-1 infection. The clinical implications of sub-clinical P. falciparum infection among HIV-1 infected persons warrant additional study.

A carbamate-based approach to primaquine prodrugs: antimalarial activity, chemical stability and enzymatic activation.

O-Alkyl and O-aryl carbamate derivatives of the antimalarial drug primaquine were synthesised as potential prodrugs that prevent oxidative deamination to the inactive metabolite carboxyprimaquine. Both O-alkyl and O-aryl carbamates undergo hydrolysis in alkaline and pH 7.4 phosphate buffers to the parent drug, with O-aryl carbamates being ca. 10(6)-10(10) more reactive than their O-alkyl counterparts. In human plasma O-alkyl carbamates were stable, whereas in contrast their O-aryl counterparts rapidly released the corresponding phenol product, with primaquine being released only slowly over longer incubation periods. Activation of the O-aryl carbamates in human plasma appears to be catalysed by butyrylcholinesterase (BuChE), which leads to carbamoylation of the catalytic serine of the enzyme followed by subsequent slow enzyme reactivation and release of parent drug. Most of the O-aryl and O-alkyl carbamates are activated in rat liver homogenates with half-lives ranging from 9 to 15 h, while the 4-nitrophenyl carbamate was hydrolysed too rapidly to determine an accurate rate constant. Antimalarial activity was studied using a model consisting of Plasmodium berghei, Balb C mice and Anopheles stephensi mosquitoes. When compared to controls, ethyl and n-hexyl carbamates were able to significantly reduce the percentage of infected mosquitos as well as the mean number of oocysts per infected mosquito, thus indicating that O-alkyl carbamates of primaquine have the potential to be developed as transmission-blocking antimalarial agents.

Prevalence and risk factors of Plasmodium falciparum infections in pregnant women of Luanda, Angola.

Pregnant women are at increased risk of malaria, but in Angola, epidemiologic data from this group is almost inexistent. We conducted a cross-sectional study to determine the prevalence and risk factors of Plasmodium falciparum infections in 567 pregnant Angolan women living in Luanda province. One in five women had P. falciparum at delivery, diagnosed by PCR assay. Age, residence and history of malaria during pregnancy were significantly associated with P. falciparum infection, but gravidity and use of anti-malarial drugs were not. Placental infections were significantly more common in women ≤18 years old and in primigravidae, but we could not correlate placental infections with poor pregnancy outcomes. These findings are relevant to malaria control policies in Luanda, Angola.

Evaluation of prevalence's of pfdhfr and pfdhps mutations in Angola.

BACKGROUND: Malaria is the major cause of morbidity and mortality in Angola. The most vulnerable groups to Plasmodium falciparum infection are pregnant women and children under five years of age. The use of an intermittent preventive treatment (IPT) with sulphadoxine/pyrimethamine (SP) in pregnant women was introduced in Angola in 2006 by the National Malaria Control Programme, and currently this strategy has been considered to be used for children malaria control. Considering the previous wide use of SP combination in Angola, together to the reported cases of SP treatment failure it is crucial the evaluation of the prevalence of five mutations in pfdhfr and pfdhps genes associated to P. falciparum resistance to SP before the introduction of S/P IPT in children. METHODS: The study was conducted in five provinces, with different transmission intensities: Huambo, Cabinda, Uíge, Kwanza Norte, and Malanje. The detection of the mutations in pfdhfr and pfdhps genes was carried out in 452 P. falciparum blood samples by PCR RFLP. RESULTS: For pfdhfr gene, 90,3% of the samples carried the mutation 51I, with 7.5% of mixed infections; 51% carried wild type allele 59C, with 29.2% mixed infections and; 99.1% of isolates harboured the mutant allele 108N. Concerning, pfdhps gene, 83,1% were mutant type 437G with 11% mixed infections , while 87% of the studied isolates were wild type for codon 540. DISCUSSION: This is the first representative epidemiological study of the whole Angola country on the prevalence of the genotypes associated with SP chemoresistance. A high frequency of individual mutations in both genes (51I and 108N in pfdhfr, and 437G in pfdhps) was found, besides a low prevalence of the quintuple mutation. CONCLUSION: The data showed that the implementation IPT using SP in children needs to be reviewed.

Molecular markers of antifolate resistance in Plasmodium falciparum isolates from Luanda, Angola.

BACKGROUND: Plasmodium falciparum malaria remains a leading health problem in Africa and its control is seriously challenged by drug resistance. Although resistance to the sulphadoxine-pyrimethamine (SP) is widespread, this combination remains an important component of malaria control programmes as intermittent preventive therapy (IPT) for pregnant women and children. In Angola, resistance patterns have been poorly characterized, and IPT has been employed for pregnant women since 2006. The aim of this study was to assess the prevalence of key antifolate resistance mediating polymorphisms in the pfdhfr and pfdhps genes in P. falciparum samples from Angola. METHODS: Plasmodium falciparum samples collected in Luanda, in 2007, were genotyped by amplification and DNA forward and reverse sequencing of the pfdhfr and pfdhps genes. RESULTS: The most prevalent polymorphisms identified were pfdhfr 108N (100%), 51I (93%), 59R (57%) and pfdhps 437G (93%). Resistance-mediating polymorphisms in pfdhps less commonly observed in West Africa were also identified (540E in 10%, 581G in 7% of samples). CONCLUSION: This study documents an important prevalence of 4 P. falciparum polymorphisms that predicts an antifolate resistance in Luanda. Further, some samples presented additional mutations associated to high-level resistance. These results suggest that the use of SP for IPT may no longer be warranted in Angola.

Genetic and phenotypic variation of the malaria vector Anopheles atroparvus in southern Europe.

BACKGROUND: There is a growing concern that global climate change will affect the potential for pathogen transmission by insect species that are vectors of human diseases. One of these species is the former European malaria vector, Anopheles atroparvus. Levels of population differentiation of An. atroparvus from southern Europe were characterized as a first attempt to elucidate patterns of population structure of this former malaria vector. Results are discussed in light of a hypothetical situation of re-establishment of malaria transmission. METHODS: Genetic and phenotypic variation was analysed in nine mosquito samples collected from five European countries, using eight microsatellite loci and geometric morphometrics on 21 wing landmarks. RESULTS: Levels of genetic diversity were comparable to those reported for tropical malaria vectors. Low levels of genetic (0.004

Identification of papain-like cysteine proteases from the bovine piroplasm Babesia bigemina and evolutionary relationship of piroplasms C1 family of cysteine proteases.

Papain-like cysteine proteases have been shown to have essential roles in parasitic protozoa and are under study as promising drug targets. Five genes were identified by sequence similarity search to be homologous to the cysteine protease family in the ongoing Babesia bigemina genome sequencing project database and were compared with the annotated genes from the complete bovine piroplasm genomes of Babesia bovis, Theileria annulata, and Theileria parva. Multiple genome alignments and sequence analysis were used to evaluate the molecular evolution events that occurred in the C1 family of cysteine proteases in these piroplasms of veterinary importance. BbiCPL1, one of the newly identified cysteine protease genes in the B. bigemina genome was expressed in Escherichia coli and shows activity against peptide substrates. Considerable differences were observed in the cysteine protease family between Babesia and Theileria genera, and this may partially explain the diverse infection mechanisms of these tick-borne diseases.

A review of antimalarial plants used in traditional medicine in communities in Portuguese-speaking countries: Brazil, Mozambique, Cape Verde, Guinea-Bissau, São Tomé and Príncipe and Angola.

The isolation of bioactive compounds from medicinal plants, based on traditional use or ethnomedical data, is a highly promising potential approach for identifying new and effective antimalarial drug candidates. The purpose of this review was to create a compilation of the phytochemical studies on medicinal plants used to treat malaria in traditional medicine from the Community of Portuguese-Speaking Countries (CPSC): Angola, Brazil, Cape Verde, Guinea-Bissau, Mozambique and São Tomé and Príncipe. In addition, this review aimed to show that there are several medicinal plants popularly used in these countries for which few scientific studies are available. The primary approach compared the antimalarial activity of native species used in each country with its extracts, fractions and isolated substances. In this context, data shown here could be a tool to help researchers from these regions establish a scientific and technical network on the subject for the CPSC where malaria is a public health problem.

Two nonrecombining sympatric forms of the human malaria parasite Plasmodium ovale occur globally.

BACKGROUND: Malaria in humans is caused by apicomplexan parasites belonging to 5 species of the genus Plasmodium. Infections with Plasmodium ovale are widely distributed but rarely investigated, and the resulting burden of disease is not known. Dimorphism in defined genes has led to P. ovale parasites being divided into classic and variant types. We hypothesized that these dimorphs represent distinct parasite species. METHODS: Multilocus sequence analysis of 6 genetic characters was carried out among 55 isolates from 12 African and 3 Asia-Pacific countries. RESULTS: Each genetic character displayed complete dimorphism and segregated perfectly between the 2 types. Both types were identified in samples from Ghana, Nigeria, São Tomé, Sierra Leone, and Uganda and have been described previously in Myanmar. Splitting of the 2 lineages is estimated to have occurred between 1.0 and 3.5 million years ago in hominid hosts. CONCLUSIONS: We propose that P. ovale comprises 2 nonrecombining species that are sympatric in Africa and Asia. We speculate on possible scenarios that could have led to this speciation. Furthermore, the relatively high frequency of imported cases of symptomatic P. ovale infection in the United Kingdom suggests that the morbidity caused by ovale malaria has been underestimated.

Molecular evolution of the three short PGRPs of the malaria vectors Anopheles gambiae and Anopheles arabiensis in East Africa.

BACKGROUND: Immune responses to parasites, which start with pathogen recognition, play a decisive role in the control of the infection in mosquitoes. Peptidoglycan recognition proteins (PGRPs) are an important family of pattern recognition receptors that are involved in the activation of these immune reactions. Pathogen pressure can exert adaptive changes in host genes that are crucial components of the vector's defence. The aim of this study was to determine the molecular evolution of the three short PGRPs (PGRP-S1, PGRP-S2 and PGRP-S3) in the two main African malaria vectors - Anopheles gambiae and Anopheles arabiensis. RESULTS: Genetic diversity of An. gambiae and An. arabiensis PGRP-S1, PGRP-S2 and PGRP-S3 was investigated in samples collected from Mozambique and Tanzania. PGRP-S1 diversity was lower than for PGRP-S2 and PGRP-S3. PGRP-S1 was the only gene differentiated between the two species. All the comparisons made for PGRP-S1 showed significant P-values for Fst estimates and AMOVA confirming a clear separation between species. For PGRP-S2 and PGRP-S3 genes it was not possible to group populations either by species or by geographic region. Phylogenetic networks reinforced the results obtained by the AMOVA and Fst values. The ratio of nonsynonymous substitutions (Ka)/synonymous substitutions (Ks) for the duplicate pair PGRP-S2 and PGRP-S3 was very similar and lower than 1. The 3D model of the different proteins coded by these genes showed that amino acid substitutions were concentrated at the periphery of the protein rather than at the peptidoglycan recognition site. CONCLUSIONS: PGRP-S1 is less diverse and showed higher divergence between An. gambiae and An. arabiensis regardless of geographic location. This probably relates to its location in the chromosome-X, while PGRP-S2 and PGRP-S3, located in chromosome-2L, showed signs of autosomal introgression. The two short PGRP genes located in the chromosome-2L were under purifying selection, which suggests functional constraints. Different types of selection acting on PGRP-S1 and PGRP-S2 and S3 might be related to their different function and catalytic activity.

Malaria: looking for selection signatures in the human PKLR gene region.

The genetic component of susceptibility to malaria is both complex and multigenic and the better-known protective polymorphisms are those involving erythrocyte-specific structural proteins and enzymes. In vivo and in vitro data have suggested that pyruvate kinase deficiency, which causes a nonspherocytic haemolytic anaemia, could be protective against malaria severity in humans, but this hypothesis remains to be tested. In the present study, we conducted a combined analysis of Short Tandem Repeats (STRs) and Single Nucleotide Polymorphisms (SNPs) in the pyruvate kinase-encoding gene (PKLR) and adjacent regions (chromosome 1q21) to look for malaria selective signatures in two sub-Saharan African populations from Angola and Mozambique, in several groups with different malaria infection outcome. A European population from Portugal, including a control and a pyruvate kinase-deficient group, was used for comparison. Data from STR and SNP loci spread along the PKLR gene region showed a considerably higher differentiation between African and Portuguese populations than that usually found for neutral markers. In addition, a wider region showing strong linkage disequilibrium was found in an uncomplicated malaria group, and a haplotype was found to be associated with this clinical group. Altogether, this data suggests that malaria selective pressure is acting in this genomic region.

Analysis of malaria associated genetic traits in Cabo Verde, a melting pot of European and sub Saharan settlers.

Malaria has occurred in the Cabo Verde archipelago with epidemic characteristics since its colonization. Nowadays, it occurs in Santiago Island alone and though prophylaxis is not recommended by the World Health Organization, studies have highlight the prospect of malaria becoming a serious public health problem as a result of the presence of antimalarial drug resistance associated with mutations in the parasite populations and underscore the need for tighter surveillance. Despite the presumptive weak immune status of the population, severe symptoms of malaria are not observed and many people present a subclinical course of the disease. No data on the prevalence of sickle-cell trait and red cell glucose-6-phosphate dehydrogenase deficiency (two classical genetic factors associated with resistance to severe malaria) were available for the Cabo Verde archipelago and, therefore, we studied the low morbidity from malaria in relation to the particular genetic characteristics of the human host population. We also included the analysis of the pyruvate kinase deficiency associated gene, reported as putatively associated with resistance to the disease. Allelic frequencies of the polymorphisms examined are closer to European than to African populations and no malaria selection signatures were found. No association was found between the analyzed human factors and infection but one result is of high interest: a linkage disequilibrium test revealed an association of distant loci in the PKLR gene and adjacent regions, only in non-infected individuals. This could mean a more conserved gene region selected in association to protection against the infection and/or the disease.

Potential threat of malaria epidemics in a low transmission area, as exemplified by São Tomé and Príncipe.

BACKGROUND: Plasmodium falciparum is the major cause of malaria infection in the island of São Tomé, in the Republic of São Tomé and Príncipe (STP), with an incidence of 40 - 50% before 2004. Since 2004, through the coordination of the Ministry of Health of STP and their Centro Nacional de Endemias (CNE), an integrated malaria control programme has been intensively deployed on the island of São Tomé. Malaria morbidity and mortality decreased by 95% after three years of effective intervention. In the low transmission settings, however, malaria seasonal fluctuation can be a potential problem directly related to epidemics if ongoing control measures are interrupted. Studies on a number of associated factors with malaria epidemics and the measures taken to respond to outbreaks are presented. METHODS: The integrated malaria control programme included indoor residual spraying (IRS), long-lasting insecticidal nets (LLINs), intermittent preventive therapy for pregnant women, as well as early diagnosis and prompt treatment with artemisinin-based combination therapy (ACT). Regular implementation of an island-wide IRS programme was carried out yearly in 2004-2007, and enhanced throughout the island in 2009. Malaria incidence and prevalence were estimated based on passive case detection and mass screening, respectively. Slide positivity rates were used for monitoring the beginning of a malaria epidemic or a seasonal peak. RESULTS: A steep decline of ca. 95% of malaria morbidity and mortality was observed between 2004 and 2008 with use of the combined control methods. Malaria incidence was 2.0%, 1.5%, and 3.0% for 2007, 2008, and 2009, respectively. In April 2008, a cross-sectional country-wide surveillance showed malaria prevalence of 3.5%, of which 95% cases were asymptomatic carriers. Only 50% of asymptomatic carriers were cured with ACT treatment, while 90% of the symptomatic patients were cured by ACT treatment as confirmed with a follow up study. Malaria morbidity increased by three-fold during the first half of 2009 as compared to the same period in 2008. Over this period of six months, severe malaria was also noted in all age groups and malaria mortality increased by two-fold in children less than five years old. After an emergency IRS was deployed, with increased use of LLINs, and an active search of asymptomatic carriers was followed and given complete ACT treatment, malaria incidence decreased to less than 1% in the second half of 2009. CONCLUSION: At the initial stage of the integrated malaria control programme, IRS contributed to the visible effect on the rapid reduction of malaria morbidity and mortality, while this programme highlights an urgent demand for the improvement of other measures, particularly promotion of LLINs usage, with close monitoring of asymptomatic carriers and with ACT treatment in malaria transmission hotspots. In addition, both daily reports and a regular active surveillance to prevent malaria outbreaks should be established permanently, so that a fast response to epidemics can be effectively made when necessary.