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Four airport malaria cases have been observed in the vicinity of the Roissy-Charles-de-Gaulle International Airport, Paris, France. These cases were geographically very close to each other and clustered in a short period of time during the summer of 1999. The phenotype and genotype of the Plasmodium falciparumisolates obtained from these patients were determined in order to know whether a single mosquito could have infected more than one subject. The genomic characterisation of isolates was performed using the polymorphic markers merozoite surface protein 1 (Mspl) and merozoite surface protein2 (Msp2) genes, the κ and ϖ repeats domains of cg2and the dihydrofolate reductase (DHFR) genotypes. Results showed identical genotypes for isolates 1, 2 and 4 whereas the genotype of isolate 3 differed at one locus. The molecular analysis was consistent with the hypothesis that all patients could have been bitten by the same mosquito and that patient 3, may have received a different clone and an additional species. In vitrosusceptibility data did not confirm or rule out this hypothesis because isolates had the same profile of susceptibility to the tested drugs.

The adhesion of infected red blood cells (IRBCs) to the cell lining of microvasculature is thought to play a central role in the pathogenesis of severe malaria. Individual IRBC can bind to more than one host receptor and parasites with multiple binding phenotypes may cause severe disease more frequently. However, as most clinical isolates are multiclonal, previous studies were hampered by the difficulty to distinguish whether a multiadherent phenotype was due to one or more parasite population(s). We have developed a tool, based on cytoadhesion assay and GeneScan genotyping technology, which enabled us to assess on fresh isolates the capacity of adherence of individual P. falciparumgenotypes to human receptors expressed on CHO transfected cells. The cytoadhesion to ICAM-1 and CD36 of IRBCs from uncomplicated and severe malaria attacks was evaluated using this methodology. In this preliminary series conducted in non immune travelers, IRBCs from severe malaria appeared to adhere more frequently and/or strongly to ICAM-1 and CD36 in comparison with uncomplicated cases. In addition, a majority genotype able to strongly adhere to CD36 was found more frequently in isolates from severe malaria cases. Further investigations are needed to confirm the clinical relevance of these data.

The present study was undertaken to explore the antimalarial effect of a series of dicatecholate iron chelators. They may be made more or less lipophilic by increasing or reducing the length of the R substituent on the nitrogen. In vitro activity against the W2 and 3D7 clones of Plasmodium falciparum, toxicity on Vero cells and toxicity on uninfected erythrocytes by measure of the released haemoglobin were assessed for each compound. These findings were compared with the ability of iron(III), iron(II) and ferritin to reverse the inhibitory effect of catecholates. This study shows that increased lipid solubility of catecholate iron chelators does not lead to improved antimalarial activity. However, their activity is well correlated with their interaction with iron and with their toxicity against Vero cells. This study demonstrates a potent antimalarial effect of FR160 (R = C9H19) on five different strains of P. falciparum in vitro. FR160 inhibited parasite growth with an IC50 between 0.8 and 1.5 micro M. The effects of FR160 on mammalian cells were minimal compared with those obtained with malaria parasites. FR160 acted on parasites at considerably higher rates than desferrioxamine, and at all stages of parasite growth. The drug was more effective at the late trophozoite and young schizont stages, although FR160 affected rings and schizonts as well. Ascorbic acid, a free radical scavenger, reduced the activities of FR160 and artesunate. FR160 might induce formation of free radicals, which could explain why FR160 antagonized the effects of artesunate and dihydroartemisinin.

The combinations of chloroquine plus chlorpromazine and chloroquine plus prochlorperazine were evaluated in vitro for potentiation against isolates of Plasmodium falciparum. Potentiation was observed against all chloroquine-resistant isolates. There was no potentiation against 8 of 10 chloroquine-susceptible isolates. The results indicated that the phenothiazines can potentiate chloroquine action against resistant parasites found in natural conditions.

Naturally occurring malaria, arbovirus infection and hepatitis in monkeys can be a hazard for the investigator and might interfere with the outcome of experiments.63 young adult Macaca fascicularisfrom Malaysia were screened for these infections. About 1 year after their arrival in France, parasitaemia due to Plasmodiumspp., was present in 6·4% of the animals and specific antibodies in 55·5%. 19 of 35 initially positive monkeys were tested again 2 years later. Parasitaemia was found in 1 of 4 monkeys and antibodies in 11 of 19 monkeys which were initially positive. 9 of the monkeys initially tested had low titres of antibodies to the Flavivirusgenus. All animals were negative for the hepatitis B surface antigen and anti-HBc. The prevalence of IgG antibodies against hepatitis A was 46·0%. The implications in terms of control are discussed.

The antifols, inhibitors of the dihydrofolate reductase (DHFR), such as pyrimethamine and proguanil, have been used againstPlasmodium falciparumin the areas where chloroquine resistance is widespread. This use has selected resistant strains in Southeast Asia and South America. The resistance is correlated with point mutations in precise positions of the DHFR gene. A reliable semi-nested PCR diagnosis test was established and used to determine the genotypic features of 29 isolates ofP. falciparumoriginating from Africa. The results obtained by the PCR technique were compared with those of thein vitrodrug sensitivity test. Some isolates were found to be polyclonal. Among the mutations tested, only mutations on codon 108 which generate an asparagine induce a decrease in sensitivity to pyrimethamine and cycloguanil, whereas mutation on codon 59 strengthens resistance to both antifols. No mutation on codon 16 or codon 164 was found.

A combination of chloroquine and amlodipine, a derivative of 1,4-dihydropyridine calcium channel blocker, was tested against Plasmodium falciparum in vitro and P. yoelii in mice. The dextrorotary enantiomer of amlodipine, practically devoid of calcium channel blocking action, increased chloroquine accumulation inside the infected mouse erythrocytes and potentiated chloroquine action against the resistant strains of P. falciparum in vitro and P. yoelii in mice. Unlike the racemate, the dextrorotary amlodipine was not toxic to the host animal, even at the highest dose of 250 mg/kg. No potentiating effect was noted in the chloroquine-susceptible strains of P. falciparum. The results of this study indicate that chloroquine potentiation of amlodipine is probably independent of calcium channels and that a combination therapy of the dextrorotary enantiomer of amlodipine and chloroquine might be a potentially useful therapeutic strategy against chloroquine-resistant falciparum malaria.

The efficacy of a loading dose of 20 mg of chloroquine per kg of body weight per os given at intervals during the first day was evaluated in 27 patients in Madagascar with Plasmodium falciparum malaria. The conventional regimen of 25 mg/kg over 3 days (schedule 1) was thus compared with a regimen of 30 mg/kg over 2 days (schedule 2; one dose of 10 mg/kg followed by two doses of 5 mg/kg at 6-h intervals on the first day and two doses of 5 mg/kg at 12-h intervals on the second day) in terms of their clinical and parasitological efficacies, tolerance, and drug concentration-time curves. At 24 h schedule 2 gave higher chloroquine levels in blood, which induced a more rapid decrease in parasitemia. The time required for a 50% decrease in the initial parasitemia was shorter in patients on schedule 2 (14.3 +/- 1.6 h) than it was in patients on schedule 1 (35.5 +/- 5.4 h; P less than 0.01). Moreover, negative blood smears were obtained more rapidly with schedule 2 (50.8 +/- 3.7 h) than with schedule 1 (72 +/- 8.7 h). As predicted by the drug concentration-time curve, no high, potentially toxic peak drug concentration appeared and no adverse effects were observed with the loading dose regimen (schedule 2). These findings support the idea that a loading dose of 20 mg/kg given at intervals during the first 12 h is well tolerated and can be used to obtain a more rapid decrease in parasitemia and to shorten the treatment time of uncomplicated chloroquine-susceptible falciparum malaria in the field.

In this study, we examined the potential interactions between antimalarial (chloroquine, quinine, and mefloquine) and oxidant reagents. The data indicate that their effects enhance those of one another in vitro. The viability of Plasmodium falciparum in culture was assessed by [3H]hypoxanthine incorporation during 24 h of incubation in the presence of lactoperoxidase, glucose-glucose oxidase, hydrogen peroxide, chloroquine, quinine, and mefloquine, either alone or in combination. At subinhibitory concentrations, a significant inhibition was produced by the following combinations: lactoperoxidase plus hydrogen peroxide, lactoperoxidase plus glucose-glucose oxidase, lactoperoxidase plus hydrogen peroxide or glucose-glucose oxidase plus chloroquine or quinine but not with mefloquine. Deletion of any component from the system markedly decreased the toxic effect on P. falciparum. This toxic effect was not inhibited by catalase. These results indicate that the peroxidase-hydrogen peroxide system and antimalarial drugs can potentiate each other to inhibit the growth of P. falciparum.

Anaemia during malaria is not completely understood. Fourteen cases of anti-erythrocyte autoimmunisation with anti-I specificity were investigated in a Paris hospital during a period of 2 years. The patients lived in Gabon and had not been taking antimalarial chemoprophylaxis. All had chronic malaria (strongly positive antimalarial immunofluorescent antibody tests). All the recognised causes for anti-erythrocyte autoimmunity were excluded. One possible explanation for these observations is some sort of interaction between I antigen and Plasmodium,the result of which facilitated penetration of the erythrocyte by the malarial parasite.

The viability of Plasmodium falciparum in culture was assessed by [3H]hypoxanthine incorporation during 24 h of incubation with lactoperoxidase, glucose-glucose oxidase, hydrogen peroxide, halides, or thiocyanate, alone or in combination. Synergistic inhibition was produced by the following combinations: lactoperoxidase plus hydrogen peroxide, lactoperoxidase plus glucose-glucose oxidase, and lactoperoxidase plus hydrogen peroxide plus halides or thiocyanate. These inhibitory effects were reversed by catalase and glutathione. The presence of plasmodial crisis forms inside the erythrocytes suggests that the oxygen-dependent microbicidal system of phagocytes has a killing effect.

To determine virulence factors of isolates of Plasmodium falciparum and the potential role of cytokines in cerebral malaria, 46 Malagasy patients presenting with cerebral (n = 10), severe (n = 10), and uncomplicated (n = 26) malaria were enrolled in a study. The capacity of 21 of 46 P. falciparum isolates to form rosettes in vitro and to adhere to human umbilical vein endothelial cells (HUVECs) that express intercellular adhesion molecule-1 receptors and to C32 amelanotic melanoma cells that express mainly CD36 receptors was investigated together with the effects of tumor necrosis factor alpha (TNF-alpha), granulocyte macrophage-colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-6 alone and in two-by-two combinations on the cytoadherence of infected erythrocytes to HUVECs. Plasma levels of these cytokines were also measured in the patients at admission. The percentage of rosette formation was higher for the isolates from patients with cerebral (n = 6; 19.5%) and severe (n = 6; 30.5%) malaria than for those from patients with uncomplicated malaria (n = 9; 5%) (P < 0.002). The cytoadherence properties of the isolates did not differ among the three groups whatever the target cell used, but adherence to melanoma cells was systematically higher than that to HUVECs. Adhesion to HUVECs was increased more after TNF-alpha stimulation than after GM-CSF, IL-3, or IL-6 stimulation (P < 0.01). Only the combination of TNF-alpha and IL-3 enhanced cytoadherence more than TNF-alpha used alone (P < 0.02). No difference in the modulation of cytoadherence by cytokines was found in relation to the severity of the disease. TNF-alpha and IL-6 levels in peripheral blood were higher in the patients with cerebral and severe malaria than in the patients with uncomplicated malaria (P < 0.005). Most of the patients' sera contained little or no IL-3 or GM-CSF. Our results challenge the role of intercellular adhesion molecule-1 as the principal receptor mediating the cytoadherence of P. falciparum-infected erythrocytes and contrast with data obtained in the murine model.

The disposition of amopyroquin was investigated in 10 healthy volunteers after a single 2-mg/kg (body weight) intramuscular dose of amopyroquin base. The major form of the drug in plasma and in whole blood was nonmetabolized amopyroquin, and only very low levels of its primary amine derivative were detected. After a rapid absorption phase (15 min), levels in plasma declined, following a tri-exponential model with a terminal elimination half-life of 129.6 +/- 92.5 h. The apparent volume of distribution (V/F) and the systemic clearance (CL/F) were 238 +/- 75 liters/kg and 2,063 +/- 1,159 ml/min, respectively. The renal clearance, calculated by using urine excreted during the first 48 h, was 119 +/- 99 ml/min and represented about 6% of the systemic clearance. About 1.2 and 0.2% of the amopyroquin dose was excreted in the urine during the first 48 h as nonmetabolized amopyroquin and its primary amine metabolite, respectively. Twenty-two Plasmodium falciparum malaria patients were studied after treatment with one of the following regimens of intramuscularly injected amopyroquin base: 3 mg/kg (body weight), 6 mg/kg, or 6 mg/kg followed by 3 mg/kg 24 h later. Parasitemia was cleared at day 7 in one of six, four of seven, and seven of nine patients, respectively. On the basis of this study, a regimen of 12 mg/kg (body weight) administered in two or three injections is suggested.

Antibodies directed against antigens of the asexual blood stages of Plasmodium falciparum were studied in the plasma of 29 individuals infected with P. falciparum and living in two areas of Madagascar. These plasma samples were investigated by four immunological methods: indirect fluorescence, immunoprecipitation of radiolabeled P. falciparum polypeptides, inhibition of the in vitro growth of P. falciparum, and double diffusion in a gelose plate. A multifactorial correspondence analysis of the results obtained for each sample revealed that the nature of several of the antibodies varied according to the age and place of residence of the subjects. In comparison with plasma samples from older individuals, specimens from young children had a higher immunofluorescence titer, immunoprecipitated several additional peptides (90, 110, and 118 kilodaltons), revealed more precipitation lines in the Ouchterlony plate technique, and did not inhibit the in vitro growth of P. falciparum to the same extent. Furthermore, as opposed to plasma samples from individuals living in the high central plateau, plasma samples from individuals living on the east coast of the island inhibited the penetration of erythrocytes by merozoites of one of the two studied P. falciparum strains and preferentially immunoprecipitated low-, rather than high-, molecular-weight peptides.

Chloroquine uptake by Plasmodium falciparum-infected human erythrocytes (RBC) was studied in vitro before and during culture by measuring the chloroquine gradient between the cells and medium (C/M) by high-pressure liquid chromatography. The C/M values were 5.9 +/- 2.7 (n = 23) for uninfected RBC, 13 to 34 for six chloroquine-susceptible isolates (concentration required to inhibit 50% of parasite growth, less than 100 nmol/liter) in partially infected RBC (parasitemia from 0.3 to 5%) (n = 28), and 8.4 to 4.9 for four chloroquine-resistant isolates (concentration required to inhibit 50% of parasite growth, 320 to 1,500 nmol/liter) in partially infected RBC (parasitemia from 0.4 to 5%) (n = 26). Two isolates were studied before and after adaptation to continuous culture. C/M was found to decrease (34.2 to 2.1 and 19.3 to 4.9), whereas the concentration required to inhibit 50% of parasite growth increased (35 to 1,400 and 54 to 1,500 nmol/liter), thus indicating the acquisition of chloroquine resistance. These results demonstrate that chloroquine uptake decreased in RBC in which the infective strain, initially susceptible, became resistant in culture and imply that the drug is bound to ferriprotoporphyrin IX to a lesser extent or that a parasite protein competes with ferriprotoporphyrin IX to a greater extent. We suggest that genotypic modifications in the mechanism of chloroquine uptake might occur in the parasite.

The activities of novel iron chelators, alone and in combination with chloroquine, quinine, or artemether, were evaluated in vitro against susceptible and resistant clones of Plasmodium falciparum with a semimicroassay system. N4-nonyl,N1,N8-bis(2,3-dihydroxybenzoyl) spermidine hydrobromide (compound 7) demonstrated the highest level of activity: 170 nM against a chloroquine-susceptible clone and 1 microM against a chloroquine-resistant clone (50% inhibitory concentrations). Compounds 6, 8, and 10 showed antimalarial activity with 50% inhibitory concentrations of about 1 microM. Compound 7 had no effect on the activities of chloroquine, quinine, and artemether against either clone, and compound 8 did not enhance the schizontocidal action of either chloroquine or quinine against the chloroquine-resistant clone. The incubation of compound 7 with FeCI3 suppressed or decreased the in vitro antimalarial activity of compound 7, while no effect was observed with incubation of compound 7 with CuSO4 and ZnSO4. These results suggest that iron deprivation may be the main mechanism of action of compound 7 against the malarial parasites. Chelator compounds 7 and 8 primarily affected trophozoite stages, probably by influencing the activity of ribonucleotide reductase, and thus inhibiting DNA synthesis.

The efficacy of a 12-mg/kg (of body weight) intramuscular amopyroquin (ApQ) regimen (two successive 6-mg/kg injections at a 24-h interval), previously established from kinetic studies on healthy volunteers and multicenter studies on patients with malaria, was investigated in 152 patients (children and adults) in Gabon with Plasmodium falciparum malaria. All children in the present study (ages, 1 to 14 years) showed higher degrees of parasitemia and temperatures and lower hematocrit values than did adults at the time of admission. No major side effects in the patients were observed. On day 7, all patients were apyretic; clearance of parasites was obtained in 143 of 152 patients (94%); a low level of parasitemia was observed in nine patients, all of whom were children (6%). In vitro chemosusceptibility tests carried out on P. falciparum isolates from patients demonstrated 51% of resistance to chloroquine (Cq). A correlation was found between the in vitro chemosusceptibilities to Cq and ApQ, but no relationship between the in vitro activity and the in vivo efficacy of ApQ could be found. Concentrations of ApQ in blood assayed by high-performance liquid chromatography on day 2 did not differ significantly between the groups in whom therapy was a success or a failure, although the mean ApQ concentration in blood for the group that failed therapy was 31% lower. Concentrations greater than 100 nmol of self-prescribed Cq and amodiaquine per liter, which were assayed simultaneously with ApQ, were observed in 78 patients (51%). They did not correlate with degrees of parasitemia compared with ApQ alone, which did. Successful treatment by day 7 was obtained in 69 of 74 patients (93%) who had no other 4-aminoquinolines in their blood. The results of the present study show that an ApQ regimen of 12 mg/kg over 2 days may be an alternative for the treatment of Cq-resistant malaria, at least in adult patients, in the field.

Congenital malaria (CM) has been considered to be rare, even in malaria-endemic areas but the disease can result in significant neonatal morbidity. Because of its rarity, the disease may go undiagnosed for a prolonged period in a seriously ill infant. We report the first case of Plasmodium malariae CM from a HIV mother. HIV could have facilitated the transfer of erythrocytic persistent P. malariae through the placenta to the fetus.

The variation of levels of tumor necrosis factor, granulocyte-macrophage colony-stimulating factor, gamma interferon, neopterin, and interleukin-2 receptors in plasma were monitored in 16 patients presenting with an acute Plasmodium falciparum malaria attack. Relations among cytokine levels and between cytokine levels and hematological and parasitological data were assessed.

The in vitro activities of furo[2,3b]quinoline and acridone alkaloids against Plasmodium falciparum were evaluated by an isotopic semimicrotest. A pyran ring in the furoquinoline nucleus and 2-O-pyranoglycoside and 2-nitro substituents in the acridone nucleus improved the antimalarial activities of the compounds. These findings provide a clue for further chemical modifications.

Fifty subjects with acute uncomplicated falciparum malaria were treated orally with a new micronized formulation of halofantrine. The dose given corresponded to one-half the normal dose for the standard formulation. Parasitemia cleared in all subjects within 78 h. There was recrudescence of falciparum malaria in seven subjects after day 14. The mean +/- standard deviation clearance times of parasitemia and fever were 49.0 +/- 14.2 and 24.3 +/- 13.2 h, respectively. Other clinical symptoms related to malaria cleared within the first 3 days. Pruritus occurred in two subjects, back pain occurred in one subject, and diarrhea occurred in one subject; all of these symptoms were mild. Hematological and biochemical indices were not adversely affected by treatment except in five subjects in whom minor and transitory increases in aspartate aminotransferase and alanine aminotransferase were observed. Micronized halofantrine appears to be a safe, well-tolerated, and effective treatment for acute falciparum malaria in semiimmune patients.

The in vitro activity of cycloguanil was assessed against 86 African isolates of Plasmodium falciparum by a semi-micro assay system. A bimodal distribution of susceptibility patterns was observed, with 44% of the isolates being cycloguanil susceptible. Cycloguanil alone retains a high activity against the intraerythrocytic forms of some isolates and, together with its activity against the hepatic stages, may be useful for chemoprophylaxis when combined with chloroquine.