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Abstract Background Discovering new lead compounds against malaria parasites is a crucial step to ensuring a sustainable global pipeline for effective anti-malarial drugs. As far as we know, no previous phytochemical or pharmacological investigations have been carried out on Sorindeia juglandifolia. This paper describes the results of an anti-malarial activity-driven investigation of the fruits of this Cameroonian plant. Methods Air-dried fruits were extracted by maceration using methanol. The extract was fractionated by flash chromatography followed by column chromatography over silica gel, eluting with gradients of hexane-ethyl acetate mixtures. Resulting fractions and compounds were tested in vitro against the Plasmodium falciparum chloroquine-resistant strain W2, against field isolates of P. falciparum, and against the P. falciparum recombinant cysteine protease falcipain-2. Promising fractions were assessed for acute toxicity after oral administration in mice. One of the promising isolated compounds was assessed in vivo against the rodent malaria parasite Plasmodium berghei. Results The main end-products of the activity-guided fractionation were 2,3,6-trihydroxy benzoic acid (1) and 2,3,6-trihydroxy methyl benzoate (2). Overall, nine fractions tested against P. falciparum W2 and falcipain-2 were active, with IC50 values of 2.3-11.6 μg/ml for W2, and 1.1-21.9 μg/ml for falcipain-2. Purified compounds (1) and (2) also showed inhibitory effects against P. falciparum W2 (IC50s 16.5 μM and 13.0 μM) and falcipain-2 (IC50s 35.4 and 6.1 μM). In studies of P. falciparum isolates from Cameroon, the plant fractions demonstrated IC50 values of 0.14-19.4 μg/ml and compounds (1) and (2) values of 6.3 and 36.1 μM. In vivo assessment of compound (1) showed activity against P. berghei strain B, with mean parasitaemia suppressive dose and curative dose of 44.9 mg/kg and 42.2 mg/kg, respectively. Active fractions were found to be safe in mice after oral administration of 7 g/kg body weight. Conclusions Fractions of Sorindeia juglandifolia and two compounds isolated from these fractions were active against cultured malaria parasites, the P. falciparum protease falcipain-2, and in a rodent malaria model. These results suggest that further investigation of the anti-malarial activities of natural products from S. juglandifolia will be appropriate.

Abstract Background Resistance to anti-malarial drugs is a widespread problem for control programmes for this devastating disease. Molecular tests are available for many anti-malarial drugs and are useful tools for the surveillance of drug resistance. However, the correlation of treatment outcome and molecular tests with particular parasite markers is not perfect, due in part to individuals who are able to clear genotypically drug-resistant parasites. This study aimed to identify molecular markers in the human genome that correlate with the clearance of malaria parasites after drug treatment, despite the drug resistance profile of the protozoan as predicted by molecular approaches. Methods 3721 samples from five African countries, which were known to contain genotypically drug resistant parasites, were analysed. These parasites were collected from patients who subsequently failed to clear their infection following drug treatment, as expected, but also from patients who successfully cleared their infections with drug-resistant parasites. 67 human polymorphisms (SNPs) on 17 chromosomes were analysed using Sequenom's mass spectrometry iPLEX gold platform, to identify regions of the human genome, which contribute to enhanced clearance of drug resistant parasites. Results An analysis of all data from the five countries revealed significant associations between the phenotype of ability to clear drug-resistant Plasmodium falciparum infection and human immune response loci common to all populations. Overall, three SNPs showed a significant association with clearance of drug-resistant parasites with odds ratios of 0.76 for SNP rs2706384 (95% CI 0.71-0.92, P = 0.005), 0.66 for SNP rs1805015 (95% CI 0.45-0.97, P = 0.03), and 0.67 for SNP rs1128127 (95% CI 0.45-0.99, P = 0.05), after adjustment for possible confounding factors. The first two SNPs (rs2706384 and rs1805015) are within loci involved in pro-inflammatory (interferon-gamma) and anti-inflammatory (IL-4) cytokine responses. The third locus encodes a protein involved in the degradation of misfolded proteins within the endoplasmic reticulum, and its role, if any, in the clearance phenotype is unclear. Conclusions The study showed significant association of three loci in the human genome with the ability of parasite to clear drug-resistant P. falciparum in samples taken from five countries distributed across sub-Saharan Africa. Both SNP rs2706384 and SNP1805015 have previously been reported to be associated with risk of malaria infection in African populations. The loci are involved in the Th1/Th2 balance, and the association of SNPs within these genes suggests a key role for antibody in the clearance of drug-resistant parasites. It is possible that patients able to clear drug-resistant infections have an enhanced ability to control parasite growth.

Abstract Background The efficacy of amodiaquine (AQ), sulphadoxine-pyrimethamine (SP) and the combination of SP+AQ in the treatment of Cameroonian children with clinical malaria was investigated. The prevalence of molecular markers for resistance to these drugs was studied to set the baseline for surveillance of their evolution with time. Methods Seven hundred and sixty children aged 6-59 months with uncomplicated falciparum malaria were studied in three ecologically different regions of Cameroon - Mutengene (littoral equatorial forest), Yaoundé (forest-savannah mosaic) and Garoua (guinea-savannah). Study children were randomized to receive either AQ, SP or the combination AQ+SP. Clinical outcome was classified according to WHO criteria, as either early treatment failure (ETF), late clinical failure (LCF), late parasitological failure (LPF) or adequate clinical and parasitological response (ACPR). The occurrence of mutations in pfcrt, pfmdr1, dhfr and dhps genes was studied by either RFLP or dot blot techniques and the prevalence of these mutations related to parasitological and therapeutic failures. Results After correction for the occurrence of re-infection by PCR, ACPRs on day 28 for AQ, SP and AQ+SP were 71.2%, 70.1% and 80.9%, in Garoua, 79.2%, 62.5%, and 81.9% in Mutengene, and 80.3%, 67.5% and 76.2% in Yaoundé respectively. High levels of Pfcrt 76T (87.11%) and Pfmdr1 86Y mutations (73.83%) were associated with quinoline resistance in the south compared to the north, 31.67% (76T) and 22.08% (86Y). There was a significant variation (p < 0.001) of the prevalence of the SGK haplotype between Garoua in the north (8.33%), Yaoundé (36.29%) in the savannah-forest mosaic and Mutengene (66.41%) in the South of Cameroon and a weak relation between SGK haplotype and SP failure. The 540E mutation on the dhps gene was extremely rare (0.3%) and occurred only in Mutengene while the pfmdr1 1034K and 1040D mutations were not detected in any of the three sites. Conclusion In this study the prevalence of molecular markers for quinoline and anti-folate resistances showed high levels and differed between the south and north of Cameroon. AQ, SP and AQ+SP treatments were well tolerated but with low levels of efficacy that suggested alternative treatments were needed in Cameroon since 2005.

Abstract Background The efficacy of artemisinin-based combination therapy has already been demonstrated in a number of studies all over the world, and some of them can be regarded as comparably effective. Ease of administration of anti-malarial treatments with shorter courses and fewer tablets may be key determinant of compliance. Methods Patients with uncomplicated falciparum malaria and over six months of age were recruited in Cameroon, Mali, Rwanda and Sudan. 1,384 patients were randomly assigned to receive artesunate-sulphamethoxypyrazine-pyrimethamine (AS-SMP) three-day (once daily for 3 days) regimen (N = 476) or AS-SMP 24-hour (0 h, 12 h, 24 h) regimen (N = 458) or artemether-lumefantrine (AL), the regular 6 doses regimen (N = 450). The primary objective was to demonstrate non-inferiority (using a margin of -6%) of AS-SMP 24 hours or AS-SMP three days versus AL on the PCR-corrected 28-day cure rate. Results The PCR corrected 28-day cure rate on the intention to treat (ITT) analysis population were: 96.0%(457/476) in the AS-SMP three-day group, 93.7%(429/458) in the AS-SMP 24-hour group and 92.0%(414/450) in the AL group. Likewise, the cure rates on the PP analysis population were high: 99.3%(432/437) in the AS-SMP three-day group, 99.5%(416/419) in the AS-SMP 24-hour group and 99.7(391/394)% in the AL group. Most common drug-related adverse events were gastrointestinal symptoms (such as vomiting and diarrhea) which were slightly higher in the AS-SMP 24-hour group. Conclusion AS-SMP three days or AS-SMP 24 hours are safe, are as efficacious as AL, and are well tolerated. Trial registration NCT00484900 http://www.clinicaltrials.gov.

Abstract Molecular markers for drug resistant malaria represent public health tools of great but mostly unrealized potential value. A key reason for the failure of molecular resistance markers to live up to their potential is that data on the their prevalence is scattered in disparate databases with no linkage to the clinical, in vitro and pharmacokinetic data that are needed to relate the genetic data to relevant phenotypes. The ongoing replacement of older monotherapies for malaria by new, more effective combination therapies presents an opportunity to create an open access database that brings together standardized data on molecular markers of drug resistant malaria from around the world. This paper presents a rationale for creating a global database of molecular markers for drug resistant malaria and for linking it to similar databases containing results from clinical trials of drug efficacy, in vitro studies of drug susceptibility, and pharmacokinetic studies of antimalarial drugs, in a World Antimalarial Resistance Network (WARN). This database will be a global resource, guiding the selection of first line drugs for treating uncomplicated malaria, for preventing malaria in travelers and for intermittent preventive treatment of malaria in pregnant women, infants and other vulnerable groups. Perhaps most important, a global database for molecular markers of drug resistant malaria will accelerate the identification and validation of markers for resistance to artemisinin-based combination therapies and, thereby, potentially prolong the useful therapeutic lives of these important new drugs.