Susceptibility of human Plasmodium knowlesi infections to anti-malarials.

BACKGROUND: Evidence suggests that Plasmodium knowlesi malaria in Sarawak, Malaysian Borneo remains zoonotic, meaning anti-malarial drug resistance is unlikely to have developed in the absence of drug selection pressure. Therefore, adequate response to available anti-malarial treatments is assumed. METHODS: Here the ex vivo sensitivity of human P. knowlesi isolates in Malaysian Borneo were studied, using a WHO schizont maturation assay modified to accommodate the quotidian life cycle of this parasite. The in vitro sensitivities of P. knowlesi H strain adapted from a primate infection to in vitro culture (by measuring the production of Plasmodium lactate dehydrogenase) were also examined together with some assays using Plasmodium falciparum and Plasmodium vivax. RESULTS: Plasmodium knowlesi is uniformly highly sensitive to artemisinins, variably and moderately sensitive to chloroquine, and less sensitive to mefloquine. CONCLUSIONS: Taken together with reports of clinical failures when P. knowlesi is treated with mefloquine, the data suggest that caution is required if using mefloquine in prevention or treatment of P. knowlesi infections, until further studies are undertaken.

Cytoadherence and virulence - the case of Plasmodium knowlesi malaria.

BACKGROUND: Cytoadherence of infected red blood cells to brain endothelium is causally implicated in malarial coma, one of the severe manifestations of falciparum malaria. Cytoadherence is mediated by specific binding of variant parasite antigens, expressed on the surface of infected erythrocytes, to endothelial receptors including, ICAM-1, VCAM and CD36. In fatal cases of severe falciparum malaria with coma, blood vessels in the brain are characteristically congested with infected erythrocytes. Brain sections from a fatal case of knowlesi malaria, but without coma, were similarly congested with infected erythrocytes. The objective of this study was to determine the binding phenotype of Plasmodium knowlesi infected human erythrocytes to recombinant human ICAM-1, VCAM and CD36. METHODS: Five patients with PCR-confirmed P. knowlesi malaria were recruited into the study with consent between April and August 2010. Pre-treatment venous blood was washed and cultured ex vivo to increase the proportion of schizont-infected erythrocytes. Cultured blood was seeded into Petri dishes with triplicate areas coated with ICAM-1, VCAM and CD36. Following incubation at 37°C for one hour the dishes were washed and the number of infected erythrocytes bound/mm2 to PBS control areas and to recombinant human ICAM-1 VCAM and CD36 coated areas were recorded. Each assay was performed in duplicate. Assay performance was monitored with the Plasmodium falciparum clone HB3. RESULTS: Blood samples were cultured ex vivo for up to 14.5 h (mean 11.3 ± 1.9 h) to increase the relative proportion of mature trophozoite and schizont-infected red blood cells to at least 50% (mean 65.8 ± 17.51%). Three (60%) isolates bound significantly to ICAM-1 and VCAM, one (20%) isolate bound to VCAM and none of the five bound significantly to CD36. CONCLUSIONS: Plasmodium knowlesi infected erythrocytes from human subjects bind in a specific but variable manner to the inducible endothelial receptors ICAM-1 and VCAM. Binding to the constitutively-expressed endothelial receptor CD36 was not detected. Further work will be required to define the pathological consequences of these interactions.

Molecular systematics of caeciliid caecilians (Amphibia: Gymnophiona) of the Western Ghats, India.

Together, Indian plus Seychelles caeciliid caecilian amphibians (Gymnophiona) constitute approximately 10% of the extant species of this order. A molecular phylogenetic analysis of all but one (or two) nominal species (16, in five genera) is presented based on mitochondrial (12S, 16S, cytb, cox1) and nuclear (RAG1) sequence data. Results strongly support monophyly of both Seychelles and peninsular Indian caeciliids, and their sister-group status. Within the Indian caeciliids, Indotyphlus and Gegeneophis are monophyletic sister genera. The phylogenetic position of Gegeneophis ramaswamii, Gegeneophis seshachari, and Gegeneophis carnosus are not well resolved, but all lie outside a well-supported clade of most northern Western Ghats Gegeneophis (madhavai, mhadeiensis, goaensis, danieli/nadkarnii). Most nominal species of Indian caeciliid are diagnosed by robust haplotype clades, though the systematics of G. carnosus-like forms in northern Kerala and southern Karnataka requires substantial further investigation. For the most part, Indian caeciliid species comprise narrowly distributed, allopatric taxa with low genetic diversity. Much greater geographic genetic diversity exists among populations referred to G. seshachari, such that some populations likely represent undescribed species. This, the first phylogenetic analysis of Indian caeciliids, generally provides additional support for recent increases in described species (eight since 1999), and a framework for ongoing taxonomic revision.

Artemisone uptake in Plasmodium falciparum-infected erythrocytes.

Artemisone is one of the most promising artemisinin derivatives in clinical trials. Previous studies with radiolabeled artemisinin and dihydroartemisinin have measured uptake in Plasmodium falciparum-infected erythrocytes. Uptake is much greater in infected than in uninfected erythrocytes, but the relative contributions of transport, binding, and metabolism to this process still await definition. In this study, we characterized mechanisms by which [(14)C]artemisone is taken up into uninfected and P. falciparum-infected human erythrocytes in vitro. Radiolabeled artemisone rapidly enters uninfected erythrocytes without much exceeding extracellular concentrations. Unlabeled artemisone does not compete in this process. Radiolabeled artemisone is concentrated greatly by a time- and temperature-dependent mechanism in infected erythrocytes. This uptake is abrogated by unlabeled artemisone. In addition, the uptake of artemisone into three subcellular fractions, and its distribution into these fractions, is examined as a function of parasite maturation. These data are relevant to an understanding of the mechanisms of action of this important class of drugs.

The distribution of Mekong schistosomiasis, past and future: preliminary indications from an analysis of genetic variation in the intermediate host.

Neotricula aperta is the only known intermediate host of Schistosoma mekongi which infects humans in Cambodia and the southern tip of Lao PDR. DNA-sequence data (partial rrnL, i.e., mitochondrial 16S large ribosomal-RNA gene) were obtained for 359 N. aperta snails sampled at 31 localities in Cambodia, Lao PDR and Thailand. A nested clade analysis was performed to detect and evaluate any geographical patterns in the observed variation and to identify genetic subpopulations or clades. Coalescent simulations were used to compare different historical biogeographical hypotheses for N. aperta and S. mekongi. A coalescent based method was also used to provide maximum likelihood estimates (MLEs) for effective populations sizes and historical growth and migration rates. Dates were also estimated for phylogenetic events on the gene tree reconstructed for the sampled haplotypes (e.g. the time to most recent common ancestor). N. aperta was found to be divided into two monophyletic clades, a spring-dwelling form of northern Lao PDR and a more widespread larger-river dwelling form of southern Lao PDR and Cambodia; this divergence was dated at 9.3 Ma. The populations with the largest estimated population sizes were found in the Mekong River of Lao PDR and Cambodia; these, together with those of the rivers of eastern Cambodia, appeared to have been the fastest growing populations. Dominant levels of gene-flow (migration) were apparent in a South to North direction, particularly out of seeder populations in the Cambodian Mekong River. The radiation of N. aperta into sub-clades across Cambodia and Lao PDR is dated at around 5 Ma. The findings suggest that historical events, rather than ecology, might best explain the absence of S. mekongi from most of Lao PDR. The public health implications of these findings are discussed, as are pointers for future studies and surveillance.

DNA-sequence variation among Schistosoma mekongi populations and related taxa; phylogeography and the current distribution of Asian schistosomiasis.

BACKGROUND: Schistosomiasis in humans along the lower Mekong River has proven a persistent public health problem in the region. The causative agent is the parasite Schistosoma mekongi (Trematoda: Digenea). A new transmission focus is reported, as well as the first study of genetic variation among S. mekongi populations. The aim is to confirm the identity of the species involved at each known focus of Mekong schistosomiasis transmission, to examine historical relationships among the populations and related taxa, and to provide data for use (a priori) in further studies of the origins, radiation, and future dispersal capabilities of S. mekongi. METHODOLOGY/PRINCIPAL FINDINGS: DNA sequence data are presented for four populations of S. mekongi from Cambodia and southern Laos, three of which were distinguishable at the COI (cox1) and 12S (rrnS) mitochondrial loci sampled. A phylogeny was estimated for these populations and the other members of the Schistosoma sinensium group. The study provides new DNA sequence data for three new populations and one new locus/population combination. A Bayesian approach is used to estimate divergence dates for events within the S. sinensium group and among the S. mekongi populations. CONCLUSIONS/SIGNIFICANCE: The date estimates are consistent with phylogeographical hypotheses describing a Pliocene radiation of the S. sinensium group and a mid-Pleistocene invasion of Southeast Asia by S. mekongi. The date estimates also provide Bayesian priors for future work on the evolution of S. mekongi. The public health implications of S. mekongi transmission outside the lower Mekong River are also discussed.