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.

Gene expression changes in the salivary glands of Anopheles coluzzii elicited by Plasmodium berghei infection.

BACKGROUND: Malaria is a devastating infectious disease caused by Plasmodium parasites transmitted through the bites of infected Anopheles mosquitoes. Salivary glands are the only mosquito tissue invaded by Plasmodium sporozoites, being a key stage for the effective parasite transmission, making the study of Anopheles sialome highly relevant. METHODS: RNA-sequencing was used to compare differential gene expression in salivary glands of uninfected and Plasmodium berghei-infected Anopheles coluzzii mosquitoes. RNA-seq results were validated by quantitative RT-PCR. The transmembrane glucose transporter gene AGAP007752 was selected for functional analysis by RNA interference. The effect of gene silencing on infection level was evaluated. The putative function and tertiary structure of the protein was assessed. RESULTS: RNA-seq data showed that 2588 genes were differentially expressed in mosquitoes salivary glands in response to P. berghei infection, being 1578 upregulated and 1010 downregulated. Metabolism, Immunity, Replication/Transcription/Translation, Proteolysis and Transport were the mosquito gene functional classes more affected by parasite infection. Endopeptidase coding genes were the most abundant within the differentially expressed genes in infected salivary glands (P < 0.001). Based on its putative function and expression level, the transmembrane glucose transporter gene, AGAP007752, was selected for functional analysis by RNA interference. The results demonstrated that the number of sporozoites was 44.3% lower in mosquitoes fed on infected mice after AGAPP007752 gene knockdown when compared to control (P < 0.01). CONCLUSIONS: Our hypothesis is that the protein encoded by the gene AGAPP007752 may play a role on An. coluzzii salivary glands infection by Plasmodium parasite, working as a sporozoite receptor and/or promoting a favorable environment for the capacity of sporozoites.

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.

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.

Treatment of Plasmodium chabaudi Parasites with Curcumin in Combination with Antimalarial Drugs: Drug Interactions and Implications on the Ubiquitin/Proteasome System.

Antimalarial drug resistance remains a major obstacle in malaria control. Evidence from Southeast Asia shows that resistance to artemisinin combination therapy (ACT) is inevitable. Ethnopharmacological studies have confirmed the efficacy of curcumin against Plasmodium spp. Drug interaction assays between curcumin/piperine/chloroquine and curcumin/piperine/artemisinin combinations and the potential of drug treatment to interfere with the ubiquitin proteasome system (UPS) were analyzed. In vivo efficacy of curcumin was studied in BALB/c mice infected with Plasmodium chabaudi clones resistant to chloroquine and artemisinin, and drug interactions were analyzed by isobolograms. Subtherapeutic doses of curcumin, chloroquine, and artemisinin were administered to mice, and mRNA was collected following treatment for RT-PCR analysis of genes encoding deubiquitylating enzymes (DUBs). Curcumin was found be nontoxic in BALB/c mice. The combination of curcumin/chloroquine/piperine reduced parasitemia to 37% seven days after treatment versus the control group's 65%, and an additive interaction was revealed. Curcumin/piperine/artemisinin combination did not show a favorable drug interaction in this murine model of malaria. Treatment of mice with subtherapeutic doses of the drugs resulted in a transient increase in genes encoding DUBs indicating UPS interference. If curcumin is to join the arsenal of available antimalarial drugs, future studies exploring suitable drug partners would be of interest.

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.

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.

Pyruvate kinase deficiency in sub-Saharan Africa: identification of a highly frequent missense mutation (G829A;Glu277Lys) and association with malaria.

BACKGROUND: Pyruvate kinase (PK) deficiency, causing hemolytic anemia, has been associated to malaria protection and its prevalence in sub-Saharan Africa is not known so far. This work shows the results of a study undertaken to determine PK deficiency occurrence in some sub-Saharan African countries, as well as finding a prevalent PK variant underlying this deficiency. MATERIALS AND METHODS: Blood samples of individuals from four malaria endemic countries (Mozambique, Angola, Equatorial Guinea and Sao Tome and Principe) were analyzed in order to determine PK deficiency occurrence and detect any possible high frequent PK variant mutation. The association between this mutation and malaria was ascertained through association studies involving sample groups from individuals showing different malaria infection and outcome status. RESULTS: The percentage of individuals showing a reduced PK activity in Maputo was 4.1% and the missense mutation G829A (Glu277Lys) in the PKLR gene (only identified in three individuals worldwide to date) was identified in a high frequency. Heterozygous carrier frequency was between 6.7% and 2.6%. A significant association was not detected between either PK reduced activity or allele 829A frequency and malaria infection and outcome, although the variant was more frequent among individuals with uncomplicated malaria. CONCLUSIONS: This was the first study on the occurrence of PK deficiency in several areas of Africa. A common PKLR mutation G829A (Glu277Lys) was identified. A global geographical co-distribution between malaria and high frequency of PK deficiency seems to occur suggesting that malaria may be a selective force raising the frequency of this 277Lys variant.

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.

Bayesian Latent Class Models in malaria diagnosis.

AIMS: The main focus of this study is to illustrate the importance of the statistical analysis in the evaluation of the accuracy of malaria diagnostic tests, without admitting a reference test, exploring a dataset (n=3317) collected in São Tomé and Príncipe. METHODS: Bayesian Latent Class Models (without and with constraints) are used to estimate the malaria infection prevalence, together with sensitivities, specificities, and predictive values of three diagnostic tests (RDT, Microscopy and PCR), in four subpopulations simultaneously based on a stratified analysis by age groups (< 5, ≥ 5 years old) and fever status (febrile, afebrile). RESULTS: In the afebrile individuals with at least five years old, the posterior mean of the malaria infection prevalence is 3.2% with a highest posterior density interval of [2.3-4.1]. The other three subpopulations (febrile ≥ 5 years, afebrile or febrile children less than 5 years) present a higher prevalence around 10.3% [8.8-11.7]. In afebrile children under-five years old, the sensitivity of microscopy is 50.5% [37.7-63.2]. In children under-five, the estimated sensitivities/specificities of RDT are 95.4% [90.3-99.5]/93.8% [91.6-96.0]--afebrile--and 94.1% [87.5-99.4]/97.5% [95.5-99.3]--febrile. In individuals with at least five years old are 96.0% [91.5-99.7]/98.7% [98.1-99.2]--afebrile--and 97.9% [95.3-99.8]/97.7% [96.6-98.6]--febrile. The PCR yields the most reliable results in four subpopulations. CONCLUSIONS: The utility of this RDT in the field seems to be relevant. However, in all subpopulations, data provide enough evidence to suggest caution with the positive predictive values of the RDT. Microscopy has poor sensitivity compared to the other tests, particularly, in the afebrile children less than 5 years. This type of findings reveals the danger of statistical analysis based on microscopy as a reference test. Bayesian Latent Class Models provide a powerful tool to evaluate malaria diagnostic tests, taking into account different groups of interest.

Functional genomics studies of Rhipicephalus (Boophilus) annulatus ticks in response to infection with the cattle protozoan parasite, Babesia bigemina.

Ticks are obligate haematophagous ectoparasites of wild and domestic animals as well as humans, considered to be second worldwide to mosquitoes as vectors of human diseases, but the most important vectors of disease-causing pathogens in domestic and wild animals. Babesia spp. are tick-borne pathogens that cause a disease called babesiosis in a wide range of animals and in humans. In particular, Babesia bovis and Babesia bigemina are transmitted by cattle ticks, Rhipicephalus (Boophilus) annulatus and Rhipicephalus microplus, which are considered the most important cattle ectoparasites with major economic impacts on cattle production. The objectives of this study were to identify R. annulatus genes differentially expressed in response to infection with B. bigemina. Functional analyses were conducted on selected genes by RNA interference in both R. annulatus and R. microplus ticks. Eight hundred randomly selected suppression-subtractive hybridisation library clones were sequenced and analysed. Molecular function Gene Ontology assignments showed that the obtained tick sequences encoded for proteins with different cellular functions. Differentially expressed genes with putative functions in tick-pathogen interactions were selected for validation of SSH results by real-time reverse transcription-PCR. Genes encoding for TROSPA, calreticulin, ricinusin and serum amyloid A were over-expressed in B. bigemina-infected ticks while Kunitz-type protease inhibitor 5 mRNA levels were down-regulated in infected ticks. Functional analysis of differentially expressed genes by double stranded RNA-mediated RNAi showed that under the conditions of the present study knockdown of TROSPA and serum amyloid A significantly reduced B. bigemina infection levels in R. annulatus while in R. microplus, knockdown of TROSPA, serum amyloid A and calreticulin also reduced pathogen infection levels when compared with controls. Several studies have characterised the tick-pathogen interface at the molecular level. However, to our knowledge this is the first report of functional genomics studies in R. annulatus infected with B. bigemina. The results reported here increase our understanding of the role of tick genes in Babesia infection/multiplication.

Expression and characterization of the Babesia bigemina cysteine protease BbiCPL1.

BbiCPL1 was the first papain-like cysteine protease from a piroplasm to be identified with proteolytic activity. Here we report the improved production of the active recombinant enzyme, and the biochemical characterization of this potential drug target. BbiCPL1 showed characteristic properties of its class, including hydrolysis of papain-family peptide substrates, an acidic pH optimum, requirement of a reducing environment for maximum activity, and inhibition by standard cysteine protease inhibitors such as E-64, leupeptin, ALLN and cystatin. The optimum pH for the protease activity against peptide substrates was 5.5, but enzymatic activity was observed between pH 4.0 and pH 9.0. At slightly basic pH 7.5, BbiCPL1 maintained 83% of maximum activity, suggesting a role in cytosol environment.

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.

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.

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.

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.

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.

Duffy negative antigen is no longer a barrier to Plasmodium vivax--molecular evidences from the African West Coast (Angola and Equatorial Guinea).

BACKGROUND: Plasmodium vivax shows a small prevalence in West and Central Africa due to the high prevalence of Duffy negative people. However, Duffy negative individuals infected with P. vivax have been reported in areas of high prevalence of Duffy positive people who may serve as supply of P. vivax strains able to invade Duffy negative erythrocytes. We investigated the presence of P. vivax in two West African countries, using blood samples and mosquitoes collected during two on-going studies. METHODOLOGY/FINDINGS: Blood samples from a total of 995 individuals were collected in seven villages in Angola and Equatorial Guinea, and 820 Anopheles mosquitoes were collected in Equatorial Guinea. Identification of the Plasmodium species was achieved by nested PCR amplification of the small-subunit rRNA genes; P. vivax was further characterized by csp gene analysis. Positive P. vivax-human isolates were genotyped for the Duffy blood group through the analysis of the DARC gene. Fifteen Duffy-negative individuals, 8 from Equatorial Guinea (out of 97) and 7 from Angola (out of 898), were infected with two different strains of P. vivax (VK210 and VK247). CONCLUSIONS: In this study we demonstrated that P. vivax infections were found both in humans and mosquitoes, which means that active transmission is occurring. Given the high prevalence of infection in mosquitoes, we may speculate that this hypnozoite-forming species at liver may not be detected by the peripheral blood samples analysis. Also, this is the first report of Duffy negative individuals infected with two different strains of P. vivax (VK247 and classic strains) in Angola and Equatorial Guinea. This finding reinforces the idea that this parasite is able to use receptors other than Duffy to invade erythrocytes, which may have an enormous impact in P. vivax current distribution.

Prevalence and genetic diversity of Babesia and Anaplasma species in cattle in Sudan.

Disease prevalence studies are one of the most valuable tools to demonstrate the risk or impact of certain infections in local and global economies. The data obtained in these studies contribute to develop strategies for disease control. The present study aims to provide information about the prevalence of babesiosis and anaplasmosis in the northern regions of Sudan. Blood samples from four different states of Sudan were collected from apparently healthy cattle (n=692), DNA was extracted and the prevalence of Babesia and Anaplasma species was analyzed by PCR. The results confirmed the presence of Babesia bigemina, Babesia bovis and Anaplasma marginale in cattle in northern Sudan with overall prevalence rates of 4.0%, 1.9% and 6.1%, respectively. Statistical analysis revealed that the prevalence of B. bigemina, B. bovis and A. marginale varies significantly between Sudanese states as well as in different age groups, while gender seems not to have a significant effect on the prevalence of these pathogens among Sudanese cattle. The highest prevalence for B. bigemina was found in the Aljazirah State while the highest number of A. marginale positive samples was reported in River Nile.