Plasmodium falciparum drug resistance-associated mutations in isolates from children living in endemic areas of Burkina Faso.

BACKGROUND: Artemisinin-based combinations therapy (ACT) is the current frontline curative therapy for uncomplicated malaria in Burkina Faso. Sulfadoxine-pyrimethamine (SP) is used for the preventive treatment of pregnant women (IPTp), while SP plus amodiaquine (SP-AQ) is recommended for children under five in seasonal malaria chemoprevention (SMC). This study aimed to assess the proportions of mutations in the P. falciparum multidrug-resistance 1 (Pfmdr1), P. falciparum chloroquine resistance transporter (Pfcrt), P. falciparum dihydrofolate reductase (pfdhfr), and P. falciparum dihydropteroate synthase (pfdhps), genes from isolates collected during household surveys in Burkina Faso. METHODS: Dried blood spots from Plasmodium falciparum-positive cases at three sites (Orodara, Gaoua, and Banfora) collected during the peak of transmission were analysed for mutations in Pfcrt (codons 72-76, 93, 97, 145, 218, 343, 350 and 353), Pfmdr-1 (codons 86, 184, 1034, 1042 and 1246) dhfr (codons 51, 59, 108, 164) and dhps (at codons 431, 436, 437, 540, 581, 613) genes using deep sequencing of multiplexed Polymerase chaine reaction (PCR) amplicons. RESULTS: Of the 377 samples analysed, 346 (91.7%), 369 (97.9%), 368 (97.6%), and 374 (99.2%) were successfully sequenced for Pfcrt, Pfmdr-1, dhfr, and dhps, respectively. Most of the samples had a Pfcrt wild-type allele (89.3%). The 76T mutation was below 10%. The most frequent Pfmdr-1 mutation was detected at codon 184 (Y > F, 30.9%). The single mutant genotype (NFSND) predominated (66.7%), followed by the wild-type genotype (NYSND, 30.4%). The highest dhfr mutations were observed at codon 59R (69.8%), followed by codons 51I (66.6%) and 108 N (14.7%). The double mutant genotype (ACIRSI) predominated (52.4%). For mutation in the dhps gene, the highest frequency was observed at codon 437 K (89.3%), followed by codons 436 A (61.2%), and 613 S (14.4%). The double mutant genotype (IAKKAA) and the single mutant genotype (ISKKAA) were predominant (37.7% and 37.2%, respectively). The most frequent dhfr/dhps haplotypes were the triple mutant ACIRSI/IAKKAA (23%), the wild-type ACNCSI/ISKKAA (19%) and the double mutant ACIRSI/ISKKAA (14%). A septuple mutant ACIRNI/VAKKGA was observed in 2 isolates from Gaoua (0.5%). CONCLUSION: The efficacy of ACT partner drugs and drugs used in IPTp and SMC does not appear to be affected by the low proportion of highly resistant mutants observed in this study. Continued monitoring, including molecular surveillance, is critical for decision-making on effective treatment policy in Burkina Faso.

In vitro antiplasmodial activity of some medicinal plants of Burkina Faso.

Malaria remains a major public health problem due to the emergence and spread of Plasmodium falciparum drug resistance. There is an urgent need to investigate new sources of antimalarial drugs which are more effective against Plasmodium falciparum. One of the potential sources of antimalarial drugs is traditional medicinal plants. In this work, we studied the in vitro antiplasmodial activity of chloromethylenic, methanolic, and MeOH/H2O (1/1) crude extracts and decoction obtained from eight medicinal plants collected in Burkina Faso and of total alkaloids for five plants. Extracts were evaluated in vitro for efficacy against Plasmodium falciparum strain K1, which is resistant to chloroquine, pyrimethamine and proguanil using the fluorescence-based SYBR Green I assay. The antiproliferative activity on human-derived hepatoma cell line HepG2 and Chinese hamster ovary (CHO) cells was evaluated using the 3-[4,5-dimethylthyazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) test in order to determine the selectivity index. Among the plant extracts tested for in vitro antiplasmodial activity, 16 were considered to be inactive (with IC50 > 10 µg/ml), six showed a moderate activity (5 < IC50 ≤ 10 µg/ml), and six were found to have a good in vitro activity with IC50 value ≤ 5 µg/ml. The highest antiplasmodial activity was found for extracts from: the alkaloid leaf extract and the chloromethylenic extracts of Combretum fragrans (IC50 = 3 µg/ml, IC50 = 5 µg/ml), the total alkaloids and the chloromethylenic leaf extracts of Combretum collinum (IC50 = 4 µg/ml), the MeOH/H2O leaf extract of Terminalia avicennioides (IC50 = 3.5 µg/ml), and the alkaloid leaf extract of Pavetta crassipes (IC50 = 5 µg/ml). Three other extracts showed moderate antiplasmodial activity (5 < IC50 ≤ 10 µg/ml): Terminalia avicennioides and Combretum fragrans methanolic extracts and Acacia kirkii alkaloid leaf extract (IC50 = 6.5, 9 and 10 µg/ml respectively). The Terminalia avicennioides crude MeOH/H2O (80:20 v/v) extract of the leaves was submitted to a successive liquid/liquid extraction with ethylacetate and n-butanol respectively. The extracts were investigated for in vitro antiplasmodial activity and antioxidant properties using DPPH(·), ABTS(+) and FRAP methods. The ethylacetate extract showed the best antiplasmodial activity (7 µg/ml) and the active constituent was isolated as ellagic acid by bioguided fractionation with an IC50 = 0.2 µM on Plasmodium falciparum and SI = 152. Besides, Terminalia avicennioides leaf extract and ellagic acid showed a good antioxidant activity. Our finding confirms the importance of investigating the antimalarial activity of plant species used in traditional medicine. Overall, two plants belonging to the Combretaceae family, Combretum fragrans and Combretum collinum appeared to be the best candidates and will be further investigated for their antiplasmodial properties, in order to isolate the molecules responsible for the antiplasmodial activity.

A phase 1b randomized, controlled, double-blinded dosage-escalation trial to evaluate the safety, reactogenicity and immunogenicity of an adenovirus type 35 based circumsporozoite malaria vaccine in Burkinabe healthy adults 18 to 45 years of age.

BACKGROUND: Ad35.CS.01 is a pre-erythrocytic malaria candidate vaccine. It is a codon optimized nucleotide sequence representing the P. falciparum circumsporozoite (CS) surface antigen inserted in a replication deficient Adenovirus 35 backbone. A Phase 1a trial has been conducted in the USA in naïve adults and showed that the vaccine was safe. The aim of this study is to assess the safety and immunogenicity of ascending dosages in sub Saharan Africa. METHODS: A double blind, randomized, controlled, dose escalation, phase Ib trial was conducted in a rural area of Balonghin, the Saponé health district (Burkina Faso). Forty-eight healthy adults aged 18-45 years were randomized into 4 cohorts of 12 to receive three vaccine doses (day 0, 28 and 84) of 10(9), 10(10), 5X10(10), 10(11) vp of Ad35.CS.01 or normal saline by intra muscular injection. Subjects were monitored carefully during the 14 days following each vaccination for non serious adverse events. Severe and serious adverse events were collected throughout the participant study duration (12 months from the first vaccination). Humoral and cellular immune responses were measured on study days 0, 28, 56, 84, 112 and 140. RESULTS: Of the forty-eight subjects enrolled, forty-four (91.7%) received all three scheduled vaccine doses. Local reactions, all of mild severity, occurred in thirteen (27.1%) subjects. Severe (grade 3) laboratory abnormalities occurred in five (10.4%) subjects. One serious adverse event was reported and attributed to infection judged unrelated to vaccine. The vaccine induced both antibody titers and CD8 T cells producing IFNγ and TNFα with specificity to CS while eliciting modest neutralizing antibody responses against Ad35. CONCLUSION: Study vaccine Ad35.CS.01 at four different dose levels was well-tolerated and modestly immunogenic in this population. These results suggest that Ad35.CS.01 should be further investigated for preliminary efficacy in human challenge models and as part of heterologous prime-boost vaccination strategies. TRIAL REGISTRATION: ClinicalTrials.gov NCT01018459 http://clinicaltrials.gov/ct2/show/NCT01018459.

Variation in haematological parameters in children less than five years of age with asymptomatic Plasmodium infection: implication for malaria field studies

During the season of high malaria transmission, most children are infected by Plasmodium, which targets red blood cells (RBCs), affecting haematological parameters. To describe these variations, we examined the haematological profiles of two groups of children living in a malaria-endemic area. A cross-sectional survey was conducted at the peak of the malaria transmission season in a rural area of Burkina Faso. After informed consent and clinical examination, blood samples were obtained from the participants for malaria diagnosis and a full blood count. Of the 414 children included in the analysis, 192 were not infected with Plasmodium, whereas 222 were asymptomatic carriers of Plasmodium infection. The mean age of the infected children was 41.8 months (range of 26.4-57.2) compared to 38.8 months (range of 22.4-55.2) for the control group (p = 0.06). The asymptomatic infected children tended to have a significantly lower mean haemoglobin level (10.8 g/dL vs. 10.4 g/dL; p < 0.001), mean lymphocyte count (4592/µL vs. 5141/µL; p = 0.004), mean platelet count (266 x 103/µL vs. 385 x 103/µL; p < 0.001) and mean RBC count (4.388 x 106/µL vs. 4.158 x 106/µL; p < 0.001) and a higher mean monocyte count (1403/µL vs. 1192/µL; p < 0.001) compared to the control group. Special attention should be applied when interpreting haematological parameters and evaluating immune responses in asymptomatic infected children living in malaria-endemic areas and enrolled in vaccine trials.

A controlled, parallel, cluster-randomized trial of community-wide screening and treatment of asymptomatic carriers of Plasmodium falciparum in Burkina Faso.

BACKGROUND: In malaria-endemic countries, large proportions of infected individuals are asymptomatic, constituting a reservoir of parasites for infection of newly hatched mosquitoes. This study evaluated the impact of screening and treatment of asymptomatic carriers of Plasmodium falciparum. METHODS: Eighteen villages were randomized (1:1) to study arms and inhabitants participated in four community screening campaigns: three before the rainy season ~1 month apart, and the fourth after the rains at ~12 months. On day 1 of campaigns 1-3, asymptomatic carriers in the intervention arm were identified by rapid diagnostic test and treated with artemether-lumefantrine. Outcomes were symptomatic malaria with parasite density >5,000/µL per person-year in children < 5 years and change in haemoglobin between days 1 and 28 of campaign 1. RESULTS: At 12 months, the number of symptomatic malaria episodes with a parasite density >5,000/µL per person-year in children < 5 years was not significantly different between arms (1.69 vs 1.60, p = 0.3482). Mean haemoglobin change in asymptomatic carriers during campaign 1 was greater in the intervention vs control arm (+0.53 g/dL vs -0.21 g/dL, p < 0.0001). ANCOVA demonstrated that mean asymptomatic carriage at the cluster level was lower in the intervention vs control arm at day 1 of campaigns 2 (5.0% vs 34.9%, p < 0.0001) and 3 (3.5% vs 31.5%, p < 0.0001), but showed only a small difference at day 1 of campaign 4 (34.6% vs 37.6%, p = 0.2982). Mean gametocyte carriage was lower in the intervention vs control arm at day 1 of campaigns 2 and 3 (0.7% vs 5.4%, p < 0.0001; 0.5% vs 5.8%, p < 0.0001), but was similar at day 1 of campaign 4 (4.9% vs 5.1%, p = 0.7208). CONCLUSIONS: Systematic screening and treatment of asymptomatic carriers at the community level did not reduce clinical malaria incidence in the subsequent transmission season, indicating greater levels of parasite clearance are required to achieve a sustained impact in this setting.

Antiplasmodial activity and toxicity of crude extracts from alternatives parts of plants widely used for the treatment of malaria in Burkina Faso: contribution for their preservation.

In order to prevent the destruction of the ecology and to sustain the flora mainly for medicinal plants, we investigated on alternative parts taken from four plants already known to display antiplasmodial activities and largely used by traditional healers in sub-Saharan Africa. The evaluated parts are bark of trunk for Zanthoxylum zanthoxyloides and leaves for Sarcocephalus latifolius instead of roots, and leaves for Combretum molle and Anogeissus leiocarpus instead of stem bark. The antiplasmodial activity of extracts of these plants was evaluated in vitro using the multi-resistant strain (W2) of Plasmodium falciparum. Antiproliferative activity was also assessed, using K562S human monocyte cell lines, along with calculation of the selectivity index (SI) of each extract. The highest in vitro antiplasmodial activity was found in the alkaloid extract of trunk bark from Z. zanthoxyloides and from the MeOH extract of A. leiocarpus leaves (IC(50) = 1.2 microg/mL and 4.9 microg/mL, respectively) with good selectivity index. Moderate activity was found in the MeOH extract (IC(50) = 5.7 microg/mL) and MeOH/H2O extract (IC(50) = 7.9 microg/mL) of C. molle leaves. Moderate activity was also found in the MeOH/H20 extract (IC(50) = 5.2 microg/mL) and the decoction (IC(50) = 8.2 microg/mL) from leaves of A. leiocarpus. No good activity was found with extracts from roots of S. latifolius. All extracts tested displayed low levels of cytotoxicity against K562S cells. The data generated clearly show that the trunk bark for Z. zanthoxyloides and the leaves for A. leiocarpus and C. molle could be used for the treatment of malaria instead of roots and stem bark.

Safety and immunogenicity of the malaria vaccine candidate MSP3 long synthetic peptide in 12-24 months-old Burkinabe children.

BACKGROUND: A Phase Ia trial in European volunteers of the candidate vaccine merozoite surface protein 3 (MSP3), a Plasmodium falciparum blood stage membrane, showed that it induces biologically active antibodies able to achieve parasite killing in vitro, while a phase Ib trial in semi-immune adult volunteers in Burkina Faso confirmed that the vaccine was safe. The aim of this study was to assess the safety and immunogenicity of this vaccine candidate in children aged 12-24 months living in malaria endemic area of Burkina Faso. METHODS: The study was a double-blind, randomized, controlled, dose escalation phase Ib trial, designed to assess the safety, reactogenicity and immunogenicity of three doses of either 15 or 30 microg of MSP3-LSP adsorbed on aluminum hydroxide in 45 children 12 to 24 months of age randomized into three equal groups. Each group received 3 vaccine doses (on days 0, 28 and 56) of either 15 microg of MSP3-LSP, 30 microg of MSP3-LSP or of the Engerix B hepatitis B vaccine. Children were visited at home daily for the 6 days following each vaccination to solicit symptoms which might be related to vaccination. Serious adverse events occurring during the study period (1 year) were recorded. Antibody responses to MSP3-LSP were measured on days 0, 28, 56 and 84. RESULTS: All 45 enrolled children received three MSP3 vaccine doses. No serious adverse events were reported. Most of the adverse events reported were mild to moderate in severity. The only reported local symptoms with grade 3 severity were swelling and induration, with an apparently dose related response. All grade 3 adverse events resolved without any sequelae. Both MSP3 doses regimens were able to elicit high levels of anti-MSP3 specific IgG1 and IgG3 antibodies in the volunteers with very little or no increase in IgG2, IgG4 and IgM classes: i.e. vaccination induced predominantly the isotypes involved in the monocyte-dependent mechanism of P. falciparum parasite-killing. CONCLUSION: Our results support the promise of MSP3-LSP as a malaria vaccine candidate, both in terms of tolerability and of immunogenicity. Further assessment of the efficacy of this vaccine is recommended. TRIAL REGISTRATION: ClinicalTrials.gov NCT00452088.