Continuous cultivation and drug susceptibility testing of Plasmodium falciparum in a malaria endemic area.

Isolates (UCH-23 and OM) and cloned strains of Plasmodium falciparum (Clones W-2 and D-6) were maintained in continuous culture for 28 to 150 days using culture media supplemented with 10% (v/v) heat inactivated semi-immune human plasma. Microscopic appearance and growth rates (R) of the parasites in media supplemented with semi-immune human plasma [R = 1.13 (W-2), 0.92 (D-6), 0.75 (OM) and 0.84 (UCH-23)] were comparable to those of parallel cultures maintained in media supplemented with 10% (v/v) heat inactivated non-immune human plasma [R = 1.42 (W-2), 0.83 (D-6), 0.66 (OM) and 0.89 (UCH-23)]. In addition, IC50 for chloroquine and mefloquine against the two cloned strains of P. falciparum maintained in culture media supplemented with either non-immune human plasma or semi-immune human plasma were identical. Although growth rates of new isolates (UCH-23 and OM) fluctuated over time, they stabilized between the 12th and 19th day of adaptation to culture. This fluctuation in growth rates of the new isolates underscores the influence of population dynamics during adaptation of P. falciparum to continuous culture. Sixty-eight percent of the primary isolates (170 of 250) obtained from patients in Ibadan were successfully adapted and maintained in continuous culture using semi-immune human plasma. The results of these studies indicate that semi-immune human plasma is a suitable supplement for continuous cultivation and drug susceptibility testing of P. falciparum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasmodium falciparum: continuous cultivation of erythrocyte stages in plasma-free culture medium

Continuous in vitro cultivation of the malaria parasite, Plasmodium falciparum, was performed in plasma-free medium. The medium used was standard RPMI 1640 supplemented with adenosine, unsaturated C-18 fatty acids, and fatty acid-free bovine serum albumin. The medium was changed daily and the cultures were diluted with washed erythrocytes twice weekly. Growth was routinely maintained for 1 month at which time the experiments were usually terminated. Although the overall growth rates were consistently lower than in control cultures with plasma, continuous growth occurred in the absence of plasma in cultures containing cis-vaccenic, oleic, and linoleic acids.     

Isolation and cultivation of Plasmodium falciparum using adult bovine serum

RPMI 1640 medium supplemented with adult bovine serum and hypoxanthine was superior to human serum-supplemented medium for the isolation of new strains of Plasmodium falciparum in Sudan. Similar observations in Indonesia have since confirmed our results. The chloroquine sensitivity of new isolates was identical in either human or bovine serum. Once acclimated to culture conditions P. falciparum strains grew better when using human serum. Erythrocyte-specific antibody present in adult bovine serum slightly inhibited merozoite invasion of uninfected cells. Removal of this cross-reactive antibody from bovine serum increased parasite multiplication to the level obtained in human serum.     

In vitro cultivation of Plasmodium falciparum: studies with modified medium supplemented with ALBUMAX II and various animal sera

RPNI, a combination of three commercially available growth media (RPMI-1640, NCTC-135 and IMDM) has been found to support long term continuous cultivation of 3D7 strain of Plasmodium falciparum in the presence of 10% bovine calf serum. During the present study, the suitability of this medium was evaluated for the development of P. falciparum in the presence of horse, goat and rabbit sera as well as various concentrations of ALBUMAX II. RPNI medium supplemented with 10% bovine calf serum (RPNI-BCS) was used as control. The cultures were maintained in candle jars protocol and parasitaemia was monitored daily up to day 7. Horse, goat and rabbit sera all supported the development of P. falciparum. Horse serum gave best results in RPNI medium and supported continuous culture up to day 100. The parasitaemia in the presence of ALBUMAX was significantly higher in RPNI than in RPMI-1640. Addition of hypoxanthine in RPMI-1640 caused an increase in parasitaemia whereas no obvious advantage could be observed in RPNI. The findings exhibited that medium RPNI has an edge over conventional RPMI-1640 medium for in vitro cultivation of P. falciparum.     

Observations on Gametogenesis in Plasmodium falciparum from Continuous Culture*

Synopsis. Gametocytes of Plasmodium falciparum were produced in continuous cultures but eventually declined in numbers after 3–4 months in vitro. Their development progressed in a consistent pattern, from small rounded, through triangular, to ellipsoidal, and finally after 8 days to crescentic forms. Morphologic maturity occurred at 8–9 days, but the gametocytes would not exflagellate in vitro, even after 14–18 days of development. Thus, current culture methods cannot produce a continuous supply of functional gametes for further studies.     

Plasmodium falciparum in culture: improved continuous flow method.

A new design of flow vessel provides a method for continuous culture of P. falciparum in a settled layer of human erythrocytes with a slow flow of culture medium over them. The parasitemia is kept fluctuating from approximately 1%, just after addition of fresh erythrocytes, to approximately 10%, 2 or 3 days later. Each vessel provides each week 3 harvests, each containing approximately 0.6--1 X 10(9) parasites.     

Conditions Favoring Gametocytogenesis in the Continuous Culture of Plasmodium falciparum1

ABSTRACT. Gametocyte formation was observed in six isolates of Plasmodium falciparum in continuous culture. Optimal conditions include constant temperature and addition of caffeine at 2.0 mM. Depletion of glucose in the medium and treatment with cAMP did not consistently trigger the formation of gametocytes.     

Helicases - feasible antimalarial drug target for Plasmodium falciparum

Of the four Plasmodium species that cause human malaria, Plasmodium falciparum is responsible for the most severe form of the disease and this parasite is developing resistance to the major antimalarial drugs. Therefore, in order to control malaria it is necessary to identify new drug targets. One feasible target might be helicases, which are important unwinding enzymes and required for almost all the nucleic acid metabolism in the malaria parasite.     

Continuous Cultivation and Drug Susceptibility Testing of Plasmodium falciparum in a Malaria Endemic Area

ABSTRACT Isolates (UCH-23 and OM) and cloned strains of Plasmodium falciparum (Clones W-2 and D-6) were maintained in continuous culture for 28 to 150 days using culture media supplemented with 10% (v/v) heat inactivated semi-immune human plasma. Microscopic appearance and growth rates (R) of the parasites in media supplemented with semi-immune human plasma [R = 1.13 (W-2), 0.92 (D-6), 0.75 (OM) and 0.84 (UCH-23)] were comparable to those of parallel cultures maintained in media supplemented with 10% (v/v) heat inactivated non-immune human plasma [R = 1.42 (W-2), 0.83 (D-6), 0.66 (OM) and 0.89 (UCH-23)]. In addition, IC50 for chloroquine and mefloquine against the two cloned strains of P. falciparum maintained in culture media supplemented with either non-immune human plasma or semi-immune human plasma were identical. Although growth rates of new isolates (UCH-23 and OM) fluctuated over time, they stabilized between the 12th and 19th day of adaptation to culture. This fluctuation in growth rates of the new isolates underscores the influence of population dynamics during adaptation of P. falciparum to continuous culture. Sixty-eight percent of the primary isolates (170 of 250) obtained from patients in Ibadan were successfully adapted and maintained in continuous culture using semi-immune human plasma. The results of these studies indicate that semi-immune human plasma is a suitable supplement for continuous cultivation and drug susceptibility testing of P. falciparum. This finding will have practical implications in malaria endemic areas where difficulties in obtaining non-immune human plasma or serum limits establishment of continuous culture of P. falciparum and its application in studies on malaria.     

Functional analysis of drug resistance in Plasmodium falciparum in the post-genomic era

Malaria has plagued humans throughout recorded history and results in the death of over 2 million people per year. The protozoan parasite Plasmodium falciparum causes the most severe form of malaria in humans. Chemotherapy has become one of the major control strategies for this parasite; however, the development of drug resistance to virtually all of the currently available drugs is causing a crisis in the use and deployment of these compounds for prophylaxis and treatment of this disease. The genome sequence of P. falciparum is providing the informational base for the use of whole-genome strategies such as bioinformatics, microarrays and genetic mapping. These approaches, together with the availability of a high-resolution genome linkage map consisting of hundreds of microsatellite markers and the advanced technologies of transfection and proteomics, will facilitate an integrated approach to address important biological questions. In this review we will discuss strategies to identify novel genes involved in the molecular mechanisms used by the parasite to circumvent the lethal effect of current chemotherapeutic agents.     

Gametocytemia in Plasmodium vivax and Plasmodium falciparum infections

Two expert research microscopists, each blinded to the other's reports, diagnosed single-species malaria infections in 2,141 adults presenting at outpatient malaria clinics in Tak Province, Thailand, and Iquitos, Peru, in May-August 1998, May-July 1999, and May-June 2001. Plasmodium vivax patients with gametocytemia had higher fever and higher parasitemia than those without gametocytemia; temperature correlated with parasitemia in the patients with gametocytemia. Plasmodium falciparum patients with gametocytemia had lower fever than those without gametocytemia, but similar parasitemia; temperature correlated with parasitemia in the patients without gametocytemia. Hematologic data in Thailand in 2001 showed lower platelet counts in P. vivax patients with gametocytemia than in the P. vivax patients without gametocytemia, whereas P. falciparum patients with gametocytemia had similar platelet counts but lower red blood cell counts, hemoglobin levels, hematocrit levels, and higher lymphocyte counts than patients without gametocytemia.     

Plasmodium falciparum: in vitro drug interaction between chloroquine and enantiomers of amlodipine

Both enantiomers of amlodipine, whose calcium antagonist action resides almost exclusively in the R(-) enantiomer, reversed chloroquine resistance in Plasmodium falciparum in vitro. R(-) enantiomer was slightly more effective than the S(+) enantiomer in potentiating chloroquine action against chloroquine-resistant strains of parasites. No potentiating effect was observed in chloroquine-sensitive parasites. Both enantiomers entered rapidly into parasitized erythrocytes to the same extent. Reversal of chloroquine resistance by the enantiomers of amlodipine was related to dose-dependent increase in the accumulation of chloroquine inside the erythrocytes parasitized by resistant parasites. These results suggest that the potentiating effect on chloroquine is independent of calcium metabolism of malaria parasites.     

Aspartic proteases of Plasmodium falciparum and other parasitic protozoa as drug targets

All parasitic protozoa contain multiple proteases, some of which are attracting attention as drug targets. Aspartic proteases are already the targets of some clinically useful drugs (e.g. chemotherapy of HIV infection) and a variety of factors make these enzymes appealing to those seeking novel antiparasite therapies. This review provides a critical analysis of the current knowledge on Plasmodium aspartic proteases termed plasmepsins, proposes a definitive nomenclature for this group of enzymes, and compares these enzymes with aspartic proteases of humans and other parasitic protozoa. The present status of attempts to obtain specific inhibitors of the parasite enzymes that will be useful as drugs is outlined and suggestions for future research priorities are proposed.