In vitro inhibition of intracellular growth of Plasmodium falciparum by immune sera

Beginning at the ring stage, synchronized cultures of Plasmodium falciparum were grown in suspension for 22-32 hours. Intracellular growth was assayed by measuring cellular uptake and incorporation into protein of 35S-methionine. Low concentrations (2%) of serum from immune humans and Aotus monkeys were found to inhibit the uptake of the 35S-methionine. The amount of inhibition for a given serum was often inversely related to its indirect fluorescent antibody test titer. Inhibition occurred during the trophozoite stage and was not obtained with a clone lacking the erythrocyte modifications referred to as knobs. Thus, a sensitive new assay is described which allows detection of factors in immune primate sera which can affect maturation of P. falciparum within the erythrocyte. These serum factors are likely to be antibodies which react with antigens expressed at the trophozoite stage on the surface of K+-infected erythrocytes.     

Non-immune human sera at higher concentrations inhibit Plasmodium falciparum growth in vitro

Plasmodium falciparum growth in vitro related to the concentration of inactivated, non-immune human serum supplement to the RPMI medium. This study investigated the concentration of non-immune serum required to support adequate in vitro parasite growth without saturating the medium. Parasitaemia was highest with 7.5% serum concentration in suspension cultures. However, peak parasitaemia obtained under static cultures with 12.5% serum concentration did not significantly differ from the level attained with suspension cultures at the same serum concentration. Ten per cent serum-supplemented medium supported parasite growth in static and suspension cultures, and levels of parasitaemia declined with further increases in serum concentration.     

In vitro growth inhibition of Plasmodium falciparum by sera from tropical splenomegaly syndrome patients

Sera from tropical splenomegaly syndrome (TSS) and non-TSS patients from the same village were examined for their ability to inhibit the in vitro growth of Plasmodium falciparum. Using synchronized malaria cultures, sera from both groups inhibited parasite development only if added before merozoite reinvasion of erythrocytes had occurred. There was no significant difference in the degree or apparent mechanism of inhibition caused by TSS and non-TSS sera. These results suggest that the aberrant immune response that results in TSS may not be associated with the elaboration of unique serum factors that differentially inhibit growth of the parasite in vitro.     

In vitro growth inhibition of Plasmodium falciparum by sera from different regions of the Philippines

Sera from different malaria endemic regions of the Republic of the Philippines were compared for their ability to inhibit growth of Plasmodium falciparum in vitro. Dialyzed serum was added to synchronous cultures containing schizonts for either the total 48 hr test period or only the last 24 hr in order to analyze the effects on erythrocytic invasion and intraerythrocytic growth, respectively. Reduction in 3H-hypoxanthine uptake was used to determine the percent of inhibition compared to nonimmune serum. One hundred seventy sera from Mindanao and Palawan in the South, the centrally located island of Mindoro, and Luzon in the North, were tested against 4 P. falciparum strains from the Philippines and 1 from Africa. Indirect fluorescent antibody titers were not predictive of inhibition. Inhibition of merozoite invasion rather than intraerythrocytic parasite growth is suggested by this study. Generally, sera were more inhibitory to parasite strains from the same geographical area than to those from more remote areas.     

Prophylactic activity of atovaquone against Plasmodium falciparum in humans.

The prophylactic antimalarial activity of atovaquone was determined in a randomized, double-blind, placebo-controlled study of healthy volunteers who were challenged by the bite of Plasmodium falciparum-infected Anopheles stephensi. Subjects were randomly assigned to one of three groups: six received seven daily doses of 750 mg of atovaquone, starting the day before challenge; six received a single dose of 250 mg of atovaquone the day before challenge; and four received placebo. Polymerase chain reaction- and culture-confirmed parasitemia developed in all four placebo recipients, but in none of the drug recipients, indicating that either of the atovaquone regimens provides effective prophylaxis (P = 0.005). However, in low-dose recipients, the drug levels by day 6.5 were profoundly subtherapeutic, indicating that parasites were eliminated prior to the establishment of erythrocytic infection. Atovaquone thus protects non-immune subjects against mosquito-transmitted falciparum malaria, and has causal prophylactic activity.     

Multiple synergistic interactions between atovaquone and antifolates against Plasmodium falciparum in vitro: a rational basis for combination therapy

The use of synergistic drug combinations for the treatment of drug-resistant malaria is a major strategy to slow the selection and spread of Plasmodium falciparum resistant strains. In order to investigate synergistic compounds, with different modes of action, as alternative candidates for combination therapy, we used standard in vitro P. falciparum cultures and an established synergy testing method to define interactions among dapsone (DDS), atovaquone (ATQ), chlorproguanil (CPG) and its triazine metabolite chlorcycloguanil (CCG). Strong synergy was observed in the combinations DDS/CCG and ATQ/CPG. Multiple combination of these drugs, DDS/CCG/CPG/ATQ also exhibited high synergy although not higher than that of either of the two drug combinations separately. The use of this triple combination DDS/CPG/ATQ, even without an increase in synergy over their double combinations, ATQ/CPG and DDS/CCG, would contribute towards slowing the selection pressure since these drugs act against different targets and would delay the selection of parasites resistant to the three drugs, extending the useful therapeutic life of these valuable compounds.     

The in vivo and in vitro sensitivity of Plasmodium falciparum to quinine

The in vivo and in vitro sensitivity of P. falciparum to quinine were studied simultaneously on 20 isolates of P. falciparum from infected patients in Rangoon and in Tharrawaddy Township. The in vivo study showed 85% sensitive and 5% resistance at RI level. The peak plasma quinine level in all the cases were above mean MIC on days 1, 3, 5 and 7. Schizont maturation was inhibited at 128 p.mol/well in 15% of the cases but the rest were at or below 64 p.mol/well in vitro test. However, no relationship was detected between the in vivo and in vitro sensitivity of quinine.     

Serotypic antigens of Plasmodium falciparum recognized by serotype-restricted inhibitory human sera

Immune sera from some Cambodian refugees contain functional serotypic antibodies that inhibit invasion of erythrocytes by the Camp strain but not by the FCR-3 strain of Plasmodium falciparum. Using a new assay, the "competitive heterologous antigen assay" (CHAA), the serotypic antibodies in a pool of three inhibitory sera were characterized by the antigens they precipitated. In the CHAA, immunoprecipitation of antigens by antibodies to common or cross-reacting antigenic determinants was blocked with excess heterologous unlabeled FCR-3 antigens before 3H-labeled Camp schizont and merozoite antigens were immunoprecipitated. The predominant Camp strain serotypic antigens revealed after electrophoresis and autoradiography were the major 195 Kd glycoprotein surface antigen (gp195) and its processed products at 150, 83, 73, and possibly 45 Kd. Additional serotypic antigens were identified at 180, 130, 65, 50, and 32 Kd. It is likely that one or more of these serotypic antigens is a target for the serotypic antibodies that inhibit invasion.     

Plasmodium falciparum in Madagascar: in vivo and in vitro sensitivity to seven drugs

The sensitivity level of Plasmodium falciparum isolates to chloroquine and the activity of six other antimalarials were studied in the different climatic zones of Madagascar in 1983. In vivo tests were done with 10 and 25 mg kg-1 of chloroquine and amodiaquine. Early recrudescence or RII resistance was observed after treatment with 10 mg kg-1 of these drugs in 34% of the cases for chloroquine and 6.5% for amodiaquine, and after the 25 mg kg-1 dose in 7% and 0% of the cases respectively. In vitro sensitivity of 84 P. falciparum isolates to seven drugs were studied with a semi-microtest. For chloroquine, 9% of the isolates had an IC50 above 250 nM, indicating resistance. In vitro activity of piperaquine was high for all isolates except two. In vitro activity of amodiaquine, dichlorquinazine, quinine, mefloquine and halofantrine was good against all isolates (maximum IC50 was 76, 92, 560, less than or equal to 20 and less than or equal to 12 nM, respectively). Correlation between the WHO standard field test and the in vitro semi-microtest was good. Resistance of P. falciparum to chloroquine was observed in the six survey areas, but the other tested drugs showed good activity. Since no cross-resistance to 4-aminoquinolines seems to exist in Madagascar, amodiaquine (the only one available at present) should be studied as an alternative to chloroquine in the prevention and treatment of falciparum malaria in this area.     

Antimalarial activity of a riboflavin analog against Plasmodium vinckei in vivo and Plasmodium falciparum in vitro

The riboflavin analog 10-(4'-chlorophenyl)-3-methylflavin was found to have significant activity against Plasmodium vinckei vinckei when administered orally and parenterally; it was active against P. falciparum in culture. It inhibited mouse erythrocyte glutathione reductase in a dose-dependent manner. When administered orally, 5-deazariboflavin was not active in vivo although it has been shown to have activity against P. falciparum in vitro.     

Pharmacodynamic interactions of amodiaquine and its major metabolite desethylamodiaquine with artemisinin, quinine and atovaquone in Plasmodium falciparum in vitro

The antimalarial in vitro activities of amodiaquine and desethylamodiaquine in combination with atovaquone, quinine and artemisinin against Plasmodium falciparum were investigated in strains F-32, FCR-3 and K-1. These parasitic strains have different sensitivity profiles to the standard antimalarial chloroquine, but all can be considered sensitive to the test drugs, representing the recommended situation for introduction of two partner drugs in combination therapy. Amodiaquine showed marked synergism when combined with each of the three partner compounds at concentration ratios between 90 and 9x10(-7), including the therapeutically relevant range. The interaction profiles of desethylamodiaquine with quinine and artemisinin also showed predominantly synergism over a wide range of concentration ratios between 70 and 9x10(-7). The responses to all combinations exhibited signs of strain-specificity, but such phenomena were usually observed outside the therapeutic range of the concentration ratios. Synergism was generally more marked with increasing EC values, i.e. at concentrations expected to be therapeutically effective and thus clinically relevant. Even trace quantities of amodiaquine were able to potentiate the activity of structurally unrelated antimalarial drugs.     

Formation of Plasmodium falciparum gametocytes in vivo and in vitro relates to transmission intensity

Plasmodium falciparum isolates were collected during a cross-sectional survey of fever cases in an endemic area of northern India, over a period of 6 months (July-December 1996). Smears of peripheral blood from 118 cases were found to contain Plasmodium falciparum. Isolates of the parasites were successfully cultured in vitro, under identical conditions, from blood samples from 58 of the smear-positives who had asexual parasitaemias of at least 0.05%. Gametocytes developed from 50 of the 58 isolates, with a variable range of gametocytaemia. An isolate was most likely to produce gametocytes and to produce high gametocytaemias (i.e. > or = 1.0%) in vitro if it had been collected when the intensity of transmission (as reflected in bloodsmear positivity) was relatively high. A young child (< 6 years) with fever was more likely to be smear-positive for the asexual and sexual stages of P. falciparum, but less likely to be seropositive for antibodies to these stages, than the older cases of fever.     

Efficacy and safety of atovaquone/proguanil compared with mefloquine for treatment of acute Plasmodium falciparum malaria in Thailand.

The increasing frequency of therapeutic failures in falciparum malaria underscores the need for novel, rapidly effective antimalarial drugs or drug combinations. Atovaquone and proguanil are blood schizonticides that demonstrate synergistic activity against multi-drug-resistant Plasmodium falciparum in vitro. In an open-label, randomized, controlled clinical trial conducted in Thailand, adult patients with acute P. falciparum malaria were randomly assigned to treatment with atovaquone and proguanil/hydrochloride (1,000 mg and 400 mg, respectively, administered orally at 24-hr intervals for three doses) or mefloquine (750 mg administered orally, followed 6 hr later by an additional 500-mg dose). Efficacy was assessed by cure rate (the percentage of patients in whom parasitemia was eliminated and did not recur during 28 days of follow-up), parasite clearance time (PCT), and fever clearance time (FCT). Safety was assessed by sequential clinical and laboratory assessments for 28 days. Atovaquone/proguanil was significantly more effective than mefloquine (cure rate 100% [79 of 79] vs. 86% [68 of 79]; P < 0.002). The atovaquone/proguanil and mefloquine treatments did not differ with respect to PCT (mean = 65 hr versus 74 hr) or FCT (mean = 59 hr versus 51 hr). Adverse events were generally typical of malaria symptoms and each occurred in < 10% of the patients in either group, with the exception of increased vomiting found in the atovaquone/proguanil group. Transient elevations of liver enzyme levels occurred more frequently in patients treated with atovaquone/proguanil than with mefloquine, but the differences were not significant and values returned to normal by day 28 in most patients. The combination of atovaquone and proguanil was well tolerated and more effective than mefloquine in the treatment of acute uncomplicated multidrug-resistant falciparum malaria in Thailand.     

Cellular and humoral inhibition of Plasmodium falciparum growth in vitro and recovery from acute malaria

Both mononuclear cell cytotoxicity and serum inhibition of Plasmodium falciparum growth in vitro were found to vary according to the stage of infection in Gambian children with clinical malaria. Cytotoxicity was displayed by mononuclear cells and serum from children with acute malaria but this form of parasite killing was more effective in children with low grade P. falciparum infections of at least 10 days duration. Parasite inhibitory antibody was not evident in sera from acutely infected children but was found in sera from children recovering from malaria and reached a peak in convalescent children when P. falciparum growth was inhibited by at least 50%. The humoral response in convalescent children was strain related, being more effective against the most recent infecting parasite strain than against other 'wild' P. falciparum isolates. In contrast, mononuclear cell cytotoxicity was not strain related; when effective, multiplication of all parasite isolates tested was retarded to the same degree. The discussion considers the role of mononuclear cell cytotoxicity in the development of protective immunity and suggests that it may be a 'front line' defense mechanism during each malaria attack.     

In vivo and in vitro studies of quinine sensitivity of Plasmodium falciparum in Thailand.

Forty-four patients with falciparum malaria were studied. Nine patients were given quinine orally at a daily dose of 1.5 gm base for a period of 14 days. The mean parasite clearance in all 9 patients was 3.3 days, and none had recrudescence in follow-up examinations for 31 days. The in vivo study of these 9 patients showed sensitivity to quinine which correlated with the in vitro test, with concentration of quinine base 2.5-5.8 microgram/ml of blood that inhibited the maturation of Plasmodium falciparum parasites. The results of the in vitro test of 35 patients showed concentrations of quinine base 2.1-5.4 microgram/ml of blood were able to inhibit the maturation of P. falciparum parasites. Therefore, these studies indicate that Plasmodium falciparum are still sensitive to quinine and quinine remains to be the drug of choice for the treatment of falciparum malaria in Thailand.     

The susceptibility of Plasmodium falciparum to sulfadoxine and pyrimethamine: correlation of in vivo and in vitro results

In 1982, 2 of 14 Plasmodium falciparum infections acquired in East Africa and diagnosed in Copenhagen were resistant to treatment with sulfadoxine plus pyrimethamine (Fansidar), while in 1983, 6 of 18 were so. The in vivo tests were supplemented by determinations of drug concentrations in serum, and 4 isolates from in vivo-sensitive cases and 6 from in vivo-resistant cases were selected for in vivo tests. These were performed in ordinary RPMI 1640 medium and in a medium with physiological p-aminobenzoic acid and folic acid concentrations. Pharmacokinetic aberrations were found to be of possible importance in only 2 of the in vivo-resistant cases. In vitro susceptibility to sulfadoxine was found to be uniformly low in all isolates. Testing with a combination of sulfadoxine and pyrimethamine in the medium with physiological concentrations of cofactors probably reflects the in vivo situation most accurately, but in all but 1 of the isolates studied in vitro the in vivo susceptibility to Fansidar would be predicted by in vitro susceptibility to pyrimethamine in either medium. The concentration of p-aminobenzoic acid in serum, quantitated by high performance liquid chromatography, was found to be subject to wide variation, and this may have implications for in vitro testing.     

In vitro blood schizonticidal activity of sera containing mefloquine‐quinine against Plasmodium falciparum

Serum samples collected at intervals from healthy volunteers, after the administration of 3 drug regimens (quinine (QN) 600 mg, mefloquine (MQ) 750 mg, and MQ 750 mg plus QN 600 mg) were investigated for their blood schizonticidal activities against K₁ strain Plasmodium falciparum in vitro . Superiority of activity was shown in the sera collected after the combination regimen. In the diluted sera of the QN regimen, a complete inhibitory effect was observed for only 24 hours, whereas the effect was sustained for 72 hours in the sera collected after MQ in either regimen (MQ alone or MQ/QN). The pattern of minimum inhibitory concentrations (MICs) of QNEq of the sera from QN alone was constant throughout a 24‐hour period, with significantly higher concentrations than that from the combination regimen (118–150 vs 21.25–73.5 μg/l). In sera collected after the combination regimen, however, the MIC gradually decreased from 0.5 until 2.5 and 4 h, and thereafter gradually returned to the same levels again during a period of 6–24 hours. The MICs of MQEq when given as MQ alone or in combination appeared constant, with a significantly higher value in the former regimen (24.4–26.8 vs 17–19.2 μg/l).     

Screening for mutations related to atovaquone/proguanil resistance in treatment failures and other imported isolates of Plasmodium falciparum in Europe

BACKGROUND: Two single-point mutations of the Plasmodium falciparum cytochrome b gene (Tyr268Asn and Tyr268Ser) were recently reported in cases of atovaquone/proguanil (Malarone) treatment failure. However, little is known about the prevalence of codon-268 mutations and their quantitative association with treatment failure. METHODS: We set out to assess the prevalence of codon-268 mutations in P. falciparum isolates imported into Europe and to quantify their association with atovaquone/proguanil treatment failure. Isolates of P. falciparum collected by the European Network on Imported Infectious Disease Surveillance between April 2000 and August 2003 were analyzed for codon-268 mutations, by use of polymerase chain reaction-restriction fragment-length polymorphism. RESULTS: We successfully screened 504 samples for the presence of either Tyr268Ser or Tyr268Asn. One case of Ser268 and no cases of Asn268 were detected. Therefore, we can be 95% confident that the prevalence of Ser268 in the European patient pool does not exceed 0.96% and that Asn268 is less frequent than 0.77%. In 58 patients treated with atovaquone/proguanil, Tyr268Ser was present in 1 of 5 patients with treatment failure but in 0 of 53 successfully treated patients. CONCLUSIONS: Tyr268Ser seems to be a sufficient, but not a necessary, cause for atovaquone/proguanil treatment failure. The prevalence of both codon-268 mutations is currently unlikely to be >1% in the European patient pool.