The ability to undergo apoptosis, previously thought to be exclusive to multicellular organisms, has been demonstrated in unicellular parasites. On the basis of an observation that Plasmodium "crisis forms" were seen in vitro after cultivation in media containing an antimalarial drug, we attempted to determine whether Plasmodium falciparum has the ability to undergo apoptosis. By use of either the apoptosis-inducer etoposide or the antimalarial chloroquine, apoptosis in Plasmodium asexual stages was evident by the observation of DNA fragmentation and disruption of transmembrane mitochondrial potential. Next, we sought to determine whether Plasmodium produces specific cysteine proteases that can induce apoptosis. We hypothesized that the 2 metacaspase-like proteins present in the Plasmodium genome contained features typical of downstream execution steps and upstream signaling pathways such caspase activation and domain recruitment. We report that one of the metacaspase genes, PF13_0289, in addition to a universally conserved catalytic cysteine and histidine dyad required for catalysis activity, contains a putative caspase recruitment domain in the N-terminal amino acid sequence. This putative P. falciparum metacaspase protein has been designated PfMCA1. Our findings offer important insights into parasite survival strategies that could open new ways for therapeutic alternatives to drug resistance.

In vitro drug susceptibility testing with the malaria parasite has been used to assess the antimalarial activities of new compounds and to monitor drug resistance in field isolates. We investigated the validity of a SYBR green I fluorescent-based assay under various culture conditions and compared the assay results to those of previously published histidine-rich protein II (HRPII) enzyme-linked immunosorbent assay (ELISA) methods. Reference strains of Plasmodium falciparum were cultured in vitro by using standard conditions in complete medium with and without phenol red before they were dispensed into 96-well plates predosed with chloroquine, mefloquine, or quinine. Following incubation, the culture supernatants were divided and the 50% inhibitory concentrations (IC 50 s) were determined by using a SYBR green I-based method and the HRPII capture ELISA method. There were no significant differences in IC 50 values when phenol red was included in the medium. The IC 50 s and the IC 90 s of the antimalarials tested by both methods were similar or identical for each of the reference strains. Fresh clinical isolates of P. falciparum collected from imported cases of malaria in Lyon, France, were tested for in vitro resistance to chloroquine and mefloquine by using the validated SYBR green I and HRPII ELISA methods. The SYBR green I-based method was able to calculate IC 50 and IC 90 values similar or identical to those calculated by the HRPII assay with fresh clinical samples without removal of white blood cells. The SYBR green I-based method for determination of drug sensitivity levels produced results comparable to those produced by other methods, showing that this method can be used routinely to conduct surveillance for drug resistance in P. falciparum with fresh or cultured parasites.Currently, in vivo drug efficacy studies are the "gold standard" for assessment of drug resistance in malaria parasites (20). While such studies are routinely conducted on a small scale, the cost associated with in vivo drug studies with the malaria parasite prohibits sustained surveillance at the required level of vigilance. Molecular marker determination has been used to identify the single-nucleotide polymorphisms commonly associated with drug resistance in malaria parasites; however, the presence of molecular markers does not always correlate with in vivo drug resistance (25). Other molecular biology methodologies have been developed to assess the flow of drug resistance markers by using known microsatellite markers, but this technology requires specialized equipment and can be quite costly (10, 26). Although microsatellite differentiation is quite powerful and informative, it would be difficult to conduct microsatellite differentiation in the field in real time.A quick and very cost-effective alternative means to determination of the level of resistance is in vitro drug susceptibility testing (4,17). Unlike the in vivo test for drug resistance, the in vitro assay is designed to assess the parasites' responses to drugs without interf...

Cerebral involvement during malaria is a complication that leads to seizure, coma, and death. The effect of new neuroprotective therapies has not yet been investigated, although cerebral malaria shares some features with neurological stroke. Erythropoietin (EPO) is one of the more promising drugs in this area. We measured the effect of EPO on the survival of mice infected with Plasmodium berghei ANKA and demonstrated that inoculations of recombinant human EPO at the beginning of the clinical manifestations of cerebral malaria protect >90% of mice from death. This drug has no effect on the course of parasitemia. The effect of EPO was not related to either the inhibition of apoptosis in the brain or the regulation of the increase and decrease of nitric oxide production in the brain and blood, respectively. Tumor necrosis factor-alpha and interferon-gamma mRNA overexpression was inhibited by EPO, and treated mice had fewer brain hemorrhages. EPO has been used in patients with chronic diseases for years, and more recently it has been used to treat acute ischemic stroke. The data presented here provide the first evidence indicating that this cytokine could be useful for the symptomatic prevention of mortality during the acute stage of cerebral malaria.

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BackgroundThe Plasmodium falciparum NA+/H+ exchanger (pfnhe1, gene PF13_0019) has recently been proposed to influence quinine (QN) susceptibility. However, its contribution to QN resistance seems to vary geographically depending on the genetic background of the parasites. Here, the role of this gene was investigated in in vitro QN susceptibility of isolates from Viet Nam.MethodNinety-eight isolates were obtained from three different regions of the Binh Phuoc and Dak Nong bordering Cambodia provinces during 2006-2008. Among these, 79 were identified as monoclonal infection and were genotyped at the microsatellite pfnhe1 ms4760 locus and in vitro QN sensitivity data were obtained for 51 isolates. Parasite growth was assessed in the field using the HRP2 immunodetection assay.ResultsSignificant associations were found between polymorphisms at pfnhe1 microsatellite ms4760 and susceptibility to QN. Isolates with two or more DNNND exhibited much lower susceptibility to QN than those harbouring zero or one DNNND repeats (median IC50 of 682 nM versus median IC50 of 300 nM; p = 0.0146) while isolates with one NHNDNHNNDDD repeat presented significantly reduced QN susceptibility than those who had two (median IC50 of 704 nM versus median IC50 of 375 nM; p < 0.01). These QNR associated genotype features were mainly due to the over representation of profile 7 among isolates (76.5%). The majority of parasites had pfcrt76T and wild-type pfmdr1 (> 95%) thus preventing analysis of associations with these mutations. Interestingly, area with the highest median QN IC50 showed also the highest percentage of isolates carrying the pfnhe1 haplotype 7.ConclusionsThe haplotype 7 which is the typical Asian profile is likely well-adapted to high drug pressure in this area and may constitute a good genetic marker to evaluate the dissemination of QNR in this part of the world.