Optical mapping of Plasmodium falciparum chromosome 2.
Detailed restriction maps of microbial genomes are a valuable resource in genome sequencing studies but are toilsome to construct by contig construction of maps derived from cloned DNA. Analysis of genomic DNA enables large stretches of the genome to be mapped and circumvents library construction and associated cloning artifacts. We used pulsed-field gel electrophoresis purified Plasmodium falciparum chromosome 2 DNA as the starting material for optical mapping, a system for making ordered restriction maps from ensembles of individual DNA molecules. DNA molecules were bound to derivatized glass surfaces, cleaved with NheI or BamHI, and imaged by digital fluorescence microscopy. Large pieces of the chromosome containing ordered DNA restriction fragments were mapped. Maps were assembled from 50 molecules producing an average contig depth of 15 molecules and high-resolution restriction maps covering the entire chromosome. Chromosome 2 was found to be 976 kb by optical mapping with NheI, and 946 kb with BamHI, which compares closely to the published size of 947 kb from large-scale sequencing. The maps were used to further verify assemblies from the plasmid library used for sequencing. Maps generated in silico from the sequence data were compared to the optical mapping data, and good correspondence was found. Such high-resolution restriction maps may become an indispensable resource for large-scale genome sequencing projects. (+info)
8-Aminoquinolines active against blood stage Plasmodium falciparum in vitro inhibit hematin polymerization.
From the Walter Reed Army Institute of Research (WRAIR) inventory, thirteen 8-aminoquinoline analogs of primaquine were selected for screening against a panel of seven Plasmodium falciparum clones and isolates. Six of the 13 8-aminoquinolines had average 50% inhibitory concentrations between 50 and 100 nM against these P. falciparum clones and were thus an order of magnitude more potent than primaquine. However, excluding chloroquine-resistant clones and isolates, these 8-aminoquinolines were all an order of magnitude less potent than chloroquine. None of the 8-aminoquinolines was cross resistant with either chloroquine or mefloquine. In contrast to the inactive primaquine prototype, 8 of the 13 8-aminoquinolines inhibited hematin polymerization more efficiently than did chloroquine. Although alkoxy or aryloxy substituents at position 5 uniquely endowed these 13 8-aminoquinolines with impressive schizontocidal activity, the structural specificity of inhibition of both parasite growth and hematin polymerization was low. (+info)
Alternative oxidase inhibitors potentiate the activity of atovaquone against Plasmodium falciparum.
Recent evidence suggests that the malaria parasite Plasmodium falciparum utilizes a branched respiratory pathway including both a cytochrome chain and an alternative oxidase. This branched respiratory pathway model has been used as a basis for examining the mechanism of action of two antimalarial agents, atovaquone and proguanil. In polarographic assays, atovaquone immediately reduced the parasite oxygen consumption rate in a concentration-dependent manner. This is consistent with its previously described role as an inhibitor of the cytochrome bc1 complex. Atovaquone maximally inhibited the rate of P. falciparum oxygen consumption by 73% +/- 10%. At all atovaquone concentrations tested, the addition of the alternative oxidase inhibitor, salicylhydroxamic acid, resulted in a further decrease in the rate of parasite oxygen consumption. At the highest concentrations of atovaquone tested, the activities of salicylhydroxamic acid and atovaquone appear to overlap, suggesting that at these concentrations, atovaquone partially inhibits the alternative oxidase as well as the cytochrome chain. Drug interaction studies with atovaquone and salicylhydroxamic acid indicate atovaquone's activity against P. falciparum in vitro is potentiated by this alternative oxidase inhibitor, with a sum fractional inhibitory concentration of 0.6. Propyl gallate, another alternative oxidase inhibitor, also potentiated atovaquone's activity, with a sum fractional inhibitory concentration of 0.7. Proguanil, which potentiates atovaquone activity in vitro and in vivo, had a small effect on parasite oxygen consumption in polarographic assays when used alone or in the presence of atovaquone or salicylhydroxamic acid. This suggests that proguanil does not potentiate atovaquone by direct inhibition of either branch of the parasite respiratory chain. (+info)
Comparison of in vivo and in vitro tests of resistance in patients treated with chloroquine in Yaounde, Cameroon.
The usefulness of an isotopic in vitro assay in the field was evaluated by comparing its results with the therapeutic response determined by the simplified WHO in vivo test in symptomatic Cameroonian patients treated with chloroquine. Of the 117 enrolled patients, 102 (87%) completed the 14-day follow-up, and 95 isolates obtained from these patients (46 children, 49 adults) yielded an interpretable in vitro test. A total of 57 of 95 patients (60%; 28 children and 29 adults) had an adequate clinical response with negative smears (n = 46) or with an asymptomatic parasitaemia (n = 11) on day 7 and/or day 14. The geometric mean 50% inhibitory concentration of the isolates obtained from these patients was 63.3 nmol/l. Late and early treatment failure was observed in 29 (30.5%) and 9 (9.5%) patients, respectively. The geometric mean 50% inhibitory concentrations of the corresponding isolates were 173 nmol/l and 302 nmol/l. Among the patients responding with late and early treatment failure, five isolates and one isolate, respectively, yielded a discordant result (in vivo resistance and in vitro sensitivity). The sensitivity, specificity, and predictive value of the in vitro test to detect chloroquine-sensitive cases was 67%, 84% and 86%, respectively. There was moderate concordance between the in vitro and in vivo tests (kappa value = 0.48). The in vitro assay agrees relatively well with the therapeutic response and excludes several host factors that influence the results of the in vivo test. However, in view of some discordant results, the in vitro test cannot substitute for in vivo data on therapeutic efficacy. The only reliable definition of "resistance" in malaria parasites is based on clinical and parasitological response in symptomatic patients, and the in vivo test provides the standard method to determine drug sensitivity or resistance as well as to guide national drug policies. (+info)
Intraerythrocytic Plasmodium falciparum expresses a high affinity facilitative hexose transporter.
Asexual stages of Plasmodium falciparum cause severe malaria and are dependent upon host glucose for energy. We have identified a glucose transporter of P. falciparum (PfHT1) and studied its function and expression during parasite development in vitro. PfHT1 is a saturable, sodium-independent, and stereospecific transporter, which is inhibited by cytochalasin B, and has a relatively high affinity for glucose (Km = 0.48 mM) when expressed in Xenopus laevis oocytes. Competition experiments with glucose analogues show that hydroxyl groups at positions C-3 and C-4 are important for ligand binding. mRNA levels for PfHT1, assessed by the quantitative technique of tandem competitive polymerase chain reaction, are highest during the small ring stages of infection and lowest in gametocytes. Confocal immunofluorescence microscopy localizes PfHT1 to the region of the parasite plasma membrane and not to host structures. These findings have implications for development of new drug targets in malaria as well as for understanding of the pathophysiology of severe infection. When hypoglycemia complicates malaria, modeling studies suggest that the high affinity of PfHT1 is likely to increase the relative proportion of glucose taken up by parasites and thereby worsen the clinical condition. (+info)
Complexity of Plasmodium falciparum infections is consistent over time and protects against clinical disease in Tanzanian children.
The complexity of Plasmodium falciparum populations in 21 children was studied in repetitive samples over 4 years in an area of Tanzania where the organism is holoendemic. Genotyping was done by a polymerase chain reaction method that targets three highly polymorphic regions of the merozoite surface protein (MSP) 1 block 2, MSP 2, and the glutamine-rich protein. Eight children were repeatedly parasitemic, 5 had scanty parasitemias, and 8 were consistently nonparasitemic. Varying numbers of genotypes were detected in the parasitemic children, but the multiplicity of infection was significantly constant within each child. The children with frequent parasitemias experienced fewer clinical episodes during the study period than those without parasitemias. There was also a tendency for children with more complex infections to experience fewer episodes. The children had consistent parasitologic profiles over the 4 years. Although few subjects were studied and the results will require confirmation, the results suggest that asymptomatic (especially polyclonal) P. falciparum infection protects against clinical disease from new infections. (+info)
HLA class II factors associated with Plasmodium falciparum merozoite surface antigen allele families.
In Plasmodium falciparum malaria, certain human leukocyte antigens (HLA) and the parasite's merozoite surface antigens 1 and 2 (MSA-1, MSA-2) have been shown to influence the course of the infection. This report is on associations of distinct HLA factors with the occurrence of particular MSA families in a group of patients with either severe or mild P. falciparum malaria in Gabon. Different distributions of HLA-DPB1 alleles were found in the 2 groups. DR *04 alleles were observed more frequently among patients with severe malaria. Several alleles of different loci were associated with distinct MSA allele families. In addition, carriers of the amino acid methionine at position 11 of the DPA1 allele were more often infected by MSA-1 K1 parasites and less frequently by MSA-1 RO33 parasites. Furthermore, associations of HLA factors with polyclonal infections were found. (+info)
Comparison of a parasite lactate dehydrogenase-based immunochromatographic antigen detection assay (OptiMAL) with microscopy for the detection of malaria parasites in human blood samples.
Microscopic examination of blood smears remains the gold standard for malaria diagnosis, but is labor-intensive and requires skilled operators. Rapid dipstick technology provides a potential alternative. A study was conducted in The Gambia to compare the performance of OptiMAL, an immunochromatographic antigen detection assay for the diagnosis of malaria using parasite lactate dehydrogenase, against standard microscopy in patients with suspected malaria. For initial diagnosis of Plasmodium falciparum, irrespective of stage, this assay had a sensitivity of 91.3%, a specificity of 92%, a positive predictive value of 87.2%, and a negative predictive value of 94.7%. The sensitivity of the test decreased markedly at parasitemias < 0.01%. This assay can be used for the diagnosis of malaria in areas where microscopy is not available and for urgent malaria diagnosis at night and at weekends, when routine laboratories are closed and when relatively inexperienced microscopists may be on duty. (+info)