Jean Bosco Ouédraogo

Multiple populations of artemisinin-resistant Plasmodium falciparum in Cambodia

We describe an analysis of genome variation in 825 P. falciparum samples from Asia and Africa that identifies an unusual pattern of parasite population structure at the epicenter of artemisinin resistance in western Cambodia. Within this relatively small geographic area, we have discovered several distinct but apparently sympatric parasite subpopulations with extremely high levels of genetic differentiation. Of particular interest are three subpopulations, all associated with clinical resistance to artemisinin, which have skewed allele frequency spectra and high levels of haplotype homozygosity, indicative of founder effects and recent population expansion. We provide a catalog of SNPs that show high levels of differentiation in the artemisinin-resistant subpopulations, including codon variants in transporter proteins and DNA mismatch repair proteins. These data provide a population-level genetic framework for investigating the biological origins of artemisinin resistance and for defining molecular markers to assist in its elimination.

Population Genetic Analysis of Plasmodium falciparum Parasites Using a Customized Illumina GoldenGate Genotyping Assay

The diversity in the Plasmodium falciparum genome can be used to explore parasite population dynamics, with practical applications to malaria control. The ability to identify the geographic origin and trace the migratory patterns of parasites with clinically important phenotypes such as drug resistance is particularly relevant. With increasing single-nucleotide polymorphism (SNP) discovery from ongoing Plasmodium genome sequencing projects, a demand for high SNP and sample throughput genotyping platforms for large-scale population genetic studies is required. Low parasitaemias and multiple clone infections present a number of challenges to genotyping P. falciparum. We addressed some of these issues using a custom 384-SNP Illumina GoldenGate assay on P. falciparum DNA from laboratory clones (long-term cultured adapted parasite clones), short-term cultured parasite isolates and clinical (non-cultured isolates) samples from East and West Africa, Southeast Asia and Oceania. Eighty percent of the SNPs (nu200a=u200a306) produced reliable genotype calls on samples containing as little as 2 ng of total genomic DNA and on whole genome amplified DNA. Analysis of artificial mixtures of laboratory clones demonstrated high genotype calling specificity and moderate sensitivity to call minor frequency alleles. Clear resolution of geographically distinct populations was demonstrated using Principal Components Analysis (PCA), and global patterns of population genetic diversity were consistent with previous reports. These results validate the utility of the platform in performing population genetic studies of P. falciparum.

Randomized comparison of amodiaquine plus sulfadoxine-pyrimethamine artemether-lumefantrine and dihydroartemisinin-piperaquine for the treatment of uncomplicated Plasmodium falciparum malaria in Burkina Faso.

BACKGROUNDnCombination antimalarial therapy is advocated to improve treatment efficacy and limit selection of drug-resistant parasites. We compared the efficacies of 3 combination regimens in Bobo-Dioulasso, Burkina Faso: amodiaquine plus sulfadoxine-pyrimethamine, which was recently shown to be highly efficacious at this site; artemether-lumefantrine, the new national first-line antimalarial regimen; and dihydroartemisinin-piperaquine (DP), a newer regimen.nnnMETHODSnWe enrolled 559 patients >or=6 months of age with uncomplicated Plasmodium falciparum malaria and randomized them to the 3 regimens. We analyzed the risk of recurrent parasitemia by day 28 and day 42, both unadjusted and adjusted by PCR methods to distinguish recrudescence and new infection.nnnRESULTSnComplete data were available for 517 (92.5%) of the enrolled subjects. Early treatment failures occurred in 5 patients treated with amodiaquine plus sulfadoxine-pyrimethamine and in 2 patients each treated with the other regimens. The day 28 risk of recurrent parasitemia, unadjusted by genotyping, was significantly higher for patients receiving artemether-lumefantrine than for patients receiving amodiaquine plus sulfadoxine-pyrimethamine (20.1% vs. 6.2%; risk difference, 13.8%; 95% confidence interval, 7.0%-20.7%) or dihydroartemisinin-piperaquine (20.1% vs. 2.2%; risk difference, 17.9%; 95% confidence interval, 11.6%-24.1%). Similar differences were seen for children <5 years of age (54% of the study population) and when outcomes were extended to 42 days. Significant differences were not seen between outcomes for patients receiving amodiaquine plus sulfadoxine-pyrimethamine and outcomes for those receiving dihydroartemisinin-piperaquine. Recrudescences were uncommon (occurring in <5% of patients) in all treatment groups. No serious adverse events were noted.nnnCONCLUSIONSnAll regimens were highly efficacious in clearing infection, but considering the risks of recurrent malaria after therapy, the amodiaquine plus sulfadoxine-pyrimethamine and dihydroartemisinin-piperaquine regimens were more efficacious than the artemether-lumefantrine regimen (the new national regimen in Burkina Faso) for the treatment of uncomplicated P. falciparum malaria.

Artemether-lumefantrine versus amodiaquine plus sulfadoxine-pyrimethamine for uncomplicated falciparum malaria in Burkina Faso: a randomised non-inferiority trial

BACKGROUNDnArtemisinin-based combination regimens are widely advocated for malarial treatment, but other effective regimens might be cheaper and more readily available. Our aim was to compare the risk of recurrent parasitaemia in patients given artemether-lumefantrine with that in those given amodiaquine plus sulfadoxine-pyrimethamine for uncomplicated malaria.nnnMETHODSnWe enrolled 521 patients aged 6 months or older with uncomplicated falciparum malaria in Bobo-Dioulasso, Burkina Faso. Patients were randomly assigned to receive standard doses of either artemether-lumefantrine (261) or amodiaquine plus sulfadoxine-pyrimethamine (260) for 3 days. Primary endpoints were the risks of treatment failure within 28 days, either unadjusted or adjusted by genotyping to distinguish recrudescence from new infection. The study is registered at controlled-trials.gov with the identifier ISRCTN54261005.nnnFINDINGSnOf enrolled patients, 478 (92%) completed the 28-day study. The risk of recurrent symptomatic malaria was lowest in the group given amodiaquine plus sulfadoxine-pyrimethamine (1.7%vs 10.2%; risk difference 8.5%; 95% CI 4.3-12.6; p=0.0001); as was the risk of recurrent parasitaemia (4.7%vs 15.1%; 10.4%; 5.1-15.6; p=0.0002). Nearly all recurrences were due to new infections. Recrudescences were four late treatment failures with artemether-lumefantrine and one early treatment failure with amodiaquine plus sulfadoxine-pyrimethamine. Both regimens were safe and well tolerated, with pruritus more common with amodiaquine plus sulfadoxine-pyrimethamine than with artemether-lumefantrine. Each regimen selected for new isolates with mutations that have been associated with decreased drug susceptibility.nnnINTERPRETATIONnAmodiaquine plus sulfadoxine-pyrimethamine was more effective than was artemether-lumefantrine for the treatment of uncomplicated malaria. For regions of Africa where amodiaquine plus sulfadoxine-pyrimethamine continues to be effective, this less expensive and more available regimen should be considered as an alternative to blanket recommendations for artemisinin-based combination treatment for malaria.

Antibiotics in ingested human blood affect the mosquito microbiota and capacity to transmit malaria.

Malaria reduction is most efficiently achieved by vector control whereby human populations at high risk of contracting and transmitting the disease are protected from mosquito bites. Here, we identify the presence of antibiotics in the blood of malaria-infected people as a new risk of increasing disease transmission. We show that antibiotics in ingested blood enhance the susceptibility of Anopheles gambiae mosquitoes to malaria infection by disturbing their gut microbiota. This effect is confirmed in a semi-natural setting by feeding mosquitoes with blood of children naturally infected with Plasmodium falciparum. Antibiotic exposure additionally increases mosquito survival and fecundity, which are known to augment vectorial capacity. These findings suggest that malaria transmission may be exacerbated in areas of high antibiotic usage, and that regions targeted by mass drug administration programs against communicable diseases may necessitate increased vector control.

Genetic variation in human HBB is associated with Plasmodium falciparum transmission

Genetic factors are known to have a role in determining susceptibility to infectious diseases, although it is unclear whether they may also influence host efficiency in transmitting pathogens. We examine variants in HBB that have been shown to be protective against malaria and test whether these are associated with the transmission of the parasite from the human host to the Anopheles vector. We conducted cross-sectional malariological surveys on 3,739 human subjects and transmission experiments involving 60 children and 6,446 mosquitoes in Burkina Faso, West Africa. Protective hemoglobins C (HbC, β6Glu→Lys) and S (β6Glu→Val) are associated with a twofold in vivo (odds ratio 2.17, 95% CI 1.57–3.01, P = 1.0 × 10−6) and a fourfold ex vivo (odds ratio 4.12, 95% CI 1.90–9.29, P = 7.0 × 10−5) increase of parasite transmission from the human host to the Anopheles vector. This provides an example of how host genetic variation may influence the transmission dynamics of an infectious disease.

Characterization of Within-Host Plasmodium falciparum Diversity Using Next-Generation Sequence Data

Our understanding of the composition of multi-clonal malarial infections and the epidemiological factors which shape their diversity remain poorly understood. Traditionally within-host diversity has been defined in terms of the multiplicity of infection (MOI) derived by PCR-based genotyping. Massively parallel, single molecule sequencing technologies now enable individual read counts to be derived on genome-wide datasets facilitating the development of new statistical approaches to describe within-host diversity. In this class of measures the F(WS) metric characterizes within-host diversity and its relationship to population level diversity. Utilizing P. falciparum field isolates from patients in West Africa we here explore the relationship between the traditional MOI and F(WS) approaches. F(WS) statistics were derived from read count data at 86,158 SNPs in 64 samples sequenced on the Illumina GA platform. MOI estimates were derived by PCR at the msp-1 and -2 loci. Significant correlations were observed between the two measures, particularly with the msp-1 locus (Pu200a=u200a5.92×10(-5)). The F(WS) metric should be more robust than the PCR-based approach owing to reduced sensitivity to potential locus-specific artifacts. Furthermore the F(WS) metric captures information on a range of parameters which influence out-crossing risk including the number of clones (MOI), their relative proportions and genetic divergence. This approach should provide novel insights into the factors which correlate with, and shape within-host diversity.

An Effective Method to Purify Plasmodium falciparum DNA Directly from Clinical Blood Samples for Whole Genome High-Throughput Sequencing

Highly parallel sequencing technologies permit cost-effective whole genome sequencing of hundreds of Plasmodium parasites. The ability to sequence clinical Plasmodium samples, extracted directly from patient blood without a culture step, presents a unique opportunity to sample the diversity of “natural” parasite populations in high resolution clinical and epidemiological studies. A major challenge to sequencing clinical Plasmodium samples is the abundance of human DNA, which may substantially reduce the yield of Plasmodium sequence. We tested a range of human white blood cell (WBC) depletion methods on P. falciparum-infected patient samples in search of a method displaying an optimal balance of WBC-removal efficacy, cost, simplicity, and applicability to low resource settings. In the first of a two-part study, combinations of three different WBC depletion methods were tested on 43 patient blood samples in Mali. A two-step combination of Lymphoprep plus Plasmodipur best fitted our requirements, although moderate variability was observed in human DNA quantity. This approach was further assessed in a larger sample of 76 patients from Burkina Faso. WBC-removal efficacy remained high (<30% human DNA in >70% samples) and lower variation was observed in human DNA quantities. In order to assess the Plasmodium sequence yield at different human DNA proportions, 59 samples with up to 60% human DNA contamination were sequenced on the Illumina Genome Analyzer platform. An average ∼40-fold coverage of the genome was observed per lane for samples with ≤30% human DNA. Even in low resource settings, using a simple two-step combination of Lymphoprep plus Plasmodipur, over 70% of clinical sample preparations should exhibit sufficiently low human DNA quantities to enable ∼40-fold sequence coverage of the P. falciparum genome using a single lane on the Illumina Genome Analyzer platform. This approach should greatly facilitate large-scale clinical and epidemiologic studies of P. falciparum.

Experimental study of the relationship between Plasmodium gametocyte density and infection success in mosquitoes; implications for the evaluation of malaria transmission-reducing interventions

The evaluation of transmission reducing interventions (TRI) to control malaria widely uses membrane feeding assays. In such assays, the intensity of Plasmodium infection in the vector might affect the measured efficacy of the candidates to block transmission. Gametocyte density in the host blood is a determinant of the infection success in the mosquito, however, uncertain estimates of parasite densities and intrinsic characteristics of the infected blood can induce variability. To reduce this variation, a feasible method is to dilute infectious blood samples. We describe the effect of diluting samples of Plasmodium-containing blood samples to allow accurate relative measures of gametocyte densities and their impact on mosquito infectivity and TRI efficacy. Natural Plasmodium falciparum samples were diluted to generate a wide range of parasite densities, and fed to Anopheles coluzzii mosquitoes. This was compared with parallel dilutions conducted on Plasmodium berghei infections. We examined how blood dilution influences the observed blocking activity of anti-Pbs28 monoclonal antibody using the P.xa0berghei/Anopheles stephensi system. In the natural species combination P.xa0falciparum/An.xa0coluzzii, blood dilution using heat-inactivated, infected blood as diluents, revealed positive near linear relationships, between gametocyte densities and oocyst loads in the range tested. A similar relationship was observed in the P.xa0berghei/An.xa0stephensi system when using a similar dilution method. In contrast, diluting infected mice blood with fresh uninfected blood dramatically increases the infectiousness. This suggests that highly infected mice blood contains inhibitory factors or reduced blood moieties, which impede infection and may in turn, lead to misinterpretation when comparing individual TRI evaluation assays. In the lab system, the transmission blocking activity of an antibody specific for Pbs28 was confirmed to be density-dependent. This highlights the need to carefully interpret evaluations of TRI candidates, regarding gametocyte densities in the P.xa0berghei/An.xa0stephensi system.

Chloroquine-resistance molecular markers (Pfcrt T76 and Pfmdr-1 Y86) and amodiaquine resistance in Burkina Faso

We investigated the relationship between the two main molecular markers for chloroquine resistance (Pfcrt T76 and Pfmdr‐1 Y86) and the clinical efficacy of amodiaquine in Burkina Faso. Before treatment, the prevalence of Pfcrt T76, Pfmdr‐1 Y86 or both mutations in the same infection was significantly higher in patients who experienced a recrudescence than in those who successfully responded to the treatment. Therefore, these two molecular markers could be useful in monitoring amodiaquine resistance, particularly in countries where this drug is used in combination with artesunate as first‐ or second‐line treatment.