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A fast, simple, easy, efficient, and inexpensive method for DNA extraction from malaria parasites collected on filter paper would be very useful for molecular surveillance. The quality and quantity of DNA are critical to molecular diagnosis and analysis. Here, we developed a simple alkali lysis method for DNA extraction from blood samples on filter paper. The results showed that 10–50 mM NaOH and deionized water all effectively isolated parasite DNA at higher parasitemia, as witnessed by successful PCR amplification, while at a parasitemia of 0.01%, the 10 mM NaOH lysis condition generated the best results. Furthermore, DNA extracted by this method was successfully used to amplify a fragment of > 2000 bp. This method successfully extracted DNA from 1 µl of blood at a parasitemia as low as 0.0001% (equivalent to 5 parasites /µl). The DNA isolated by the 10 mM NaOH lysis method was stable to yield PCR products after storage at 4 °C or − 20 °C for 12 months. These results indicate that this alkali lysis method is simple, effective, sensitive, and inexpensive for isolating stable Plasmodium DNA from dried blood spots on filter paper. • Alkali lysis method was efficient for malaria molecular research from filter paper blood samples. • The most suitable NaOH concentration was 10 mM when extraction Plasmodium DNA from low parasitemia filter paper. • The present method enables detection of low parasitemia (0.0001% parasitemia, equivalent to 5 parasites /µl) sample. • The DNA isolated by alkali lysis method is more stable when stored at − 20 °C. [ABSTRACT FROM AUTHOR]

Sulphadoxine-pyrimethamine (SP) is only used for intermittent preventive treatment during pregnancy (IPTp) in most Sub-Saharan African countries. However, there are concerns about the efficacy of IPTp-SP because of increasing resistance. Combinations of point mutations in the dhps and dhfr genes of Plasmodium falciparum are associated with SP resistance, in particular the quintuple dhfr (N51, C59, S108) − dhps (codons A437, K540) mutant. In Nanoro, Burkina Faso, filter paper samples from pregnant women at first antenatal care visit and at delivery plus samples from the general population (GP) were studied for dhfr and dhps mutations by sequencing. Quintuple mutants were present in 2 delivery and 4 GP samples. This is the first time the quintuple mutation is found in Burkina Faso and although the prevalence is still very low, emergence of the quintuple mutation could highly diminish the efficacy of IPTp-SP. Close surveillance of SP resistance mutations is therefore warranted. [ABSTRACT FROM AUTHOR]

Abstract: In Plasmodium falciparum, the causative agent of cerebral malaria, silent information regulator 2 (Sir2) has been implicated in pathogenesis through its role in var gene silencing. P. falciparum Sir2 (PfSir2) in addition to the catalytic core, has a 13 residue N-terminal and 4 residue C-terminal extension over the shorter Archaeoglobus fulgidus Sir2. In this paper, we highlight our studies aimed at understanding the kinetic mechanism of PfSir2 and the role of N- and C-terminal extensions in protein function and oligomerization. Bisubstrate kinetic analysis showed that PfSir2 exhibits a rapid equilibrium ordered sequential mechanism, with peptide binding preceding NAD+. This study also reports on surfactin as a novel Sir2 inhibitor exhibiting competitive inhibition with respect to NAD+ and uncompetitive inhibition with acetylated peptide. This inhibition pattern with surfactin provides further support for ordered binding of substrates. Surfactin was also found to be a potent inhibitor of intra-erythrocytic growth of P. falciparum with 50% inhibitory concentration in the low micromolar range. PfSir2, like the yeast homologs (yHst2 and Sir2p), is a trimer in solution. However, dissociation of trimer to monomers in the presence of NAD+ is characteristic of the parasite enzyme. Oligomerization studies on N- and/or C-terminal deletion constructs of PfSir2 highlight the role of C-terminus of the protein in mediating homotrimerization. N-terminal deletion resulted in reduced catalytic efficiency although substrate affinity was not altered in the constructs. Interestingly, deletion of both the ends relaxed NAD+ specificity. [Copyright &y& Elsevier]

The apicoplast and mitochondrion of the malaria parasite Plasmodium falciparum are important intracellular organelles and targets of several anti-malarial drugs. In recent years, our group and others have begun to piece together the metabolic pathways of these organelles, with a view to understanding their functions and identifying further anti-malarial targets. This has involved localization of putative organellar proteins using fluorescent reporter proteins such as green fluorescent protein (GFP). A major limitation to such an approach is the difficulties associated with using existing plasmids to genetically modify P. falciparum. In this paper, we present a novel series of P. falciparum transfection vectors based around the Gateway™ recombinatorial cloning system. Our system makes it considerably easier to construct fluorescent reporter fusion proteins, as well as allowing the use of two selectable markers. Using this approach, we localize proteins involved in isoprenoid biosynthesis and the posttranslational processing of apicoplast-encoded proteins to the apicoplast, and a protein putatively involved in the citric acid cycle to the mitochondrion. To confirm the localization of these proteins, we have developed a new immunofluorescence assay (IFA) protocol using antibodies specific to the apicoplast and mitochondrion. In comparison with published IFA methods, we find that ours maintains considerably better structural preservation, while still allowing sufficient antibody binding as well as preserving reporter protein fluorescence. In summary, we present two important new tools that have enabled us to characterize some of the functions of the apicoplast and mitochondrion, and which will be of use to the wider malaria research community in elucidating the localization of other P. falciparum proteins. [Copyright &y& Elsevier]