Plasmodium yoelii: identification of a gene encoding a putative ADP-ribosylation factor-1 GTPase-activating protein, PyAG1.

The PyAG1 gene, identified by the screening of a Plasmodium yoelii genomic DNA library with a rhoptry-specific Mab, encodes a protein with a zinc finger structure immediately followed by the consensus sequence of the Arf GAP catalytic site. The serum of mice immunized with the recombinant protein recognized specifically the rhoptries of the late infected erythrocytic stages. Blast analysis using the Genbank database gave the highest scores with four proteins presenting an Arf1 GAP activity. If presenting also this activity, the PyAG1 protein could be involved in the regulation of the secreted protein vesicular transport and, consequently, in the rhoptry biogenesis.

In vitro susceptibility of African isolates of Plasmodium falciparum from Gabon to pyronaridine.

The in vitro activity of pyronaridine was evaluated against 62 isolates of Plasmodium falciparum from Libreville, Gabon using an isotopic, drug susceptibility microtest and was compared with amodiaquine, chloroquine, quinine, and halofantrine activities. The mean 50% inhibitory concentration (IC50) values of the 62 isolates from Gabon to pyronaridine was 3.0 nM (95% confidence interval [CI] = 2.1-3.9). Pyronaridine was less potent against chloroquine-resistant isolates than chloroquine-susceptible isolates but more potent than chloroquine against chloroquine-resistant parasites. The cut-off value for in vitro reduced susceptibility to pyronaridine was an IC50 > 15 nM. Two isolates (3%) showed an IC50 > 15 nM. A significant positive correlation was found between the activities of pyronaridine and chloroquine (r2 = 0.26, P < 0.001), pyronaridine and quinine (r2 = 0.36, P < 0.001), pyronaridine and amodiaquine (r2 = 0.55, P < 0.001), and pyronaridine and halofantrine (r2 = 0.50, P < 0.001). This correlation suggests in vitro cross-resistance or at least in vitro cross-susceptibility, which is not necessarily predictive of cross-resistance in vivo. The present in vitro findings require comparison with those of clinical studies.

Plasmodium yoelii: identification of rhoptry proteins using monoclonal antibodies.

Thirteen monoclonal antibodies, obtained after immunization of mice with Plasmodium yoelii schizonts, were selected using immunofluorescence assay: they all presented typical fluorescence patterns of rhoptries. This antigen localization was confirmed by immunoelectron microscopy. The molecular weights of the recognized antigens are 68, 80, 105, 130 and 140 kDa as determined by immunoprecipitation and immunoblot under reducing and nonreducing conditions. These values are very similar to these of the low and high molecular weight complex components of Plasmodium falciparum. Furthermore, these antigens are soluble like P. falciparum rhoptry proteins. Interestingly, our monoclonal antibodies also reacted with two other Plasmodium species (Plasmodium berghei NKK173 strain and P. yoelii nigeriensis 798 VK strain), giving sometimes more complex labeling with apical, membranous, nuclear, or/and cytoplasmic localizations. Finally, none of the monoclonal antibodies stained the rhoptries of P. falciparum FCCE-1/Niger strain.

Synthesis and antimalarial activities of fluoroalkyl derivatives of dihydroartemisinin.

Fluoroalkyl ethers (4) of dihydroartemisinin (2) have been prepared by reaction of fluoroalkyl alcohols with dihydroartemisinin by different methods (BF3,Et2O or TMSCl catalysis or Mitsunobu reaction). Ethers 4a-d derived from primary fluoroalkyl alcohols were obtained in moderate to good yields by these methods. Ethers 4e-j have been prepared from fluoroalkyl secondary and tertiary alcohols and phenol using the Mitsunobu reaction. Although in vitro antimalarial activities of ethers toward Plasmodium falciparum W-2 asiatic strain are moderate, in vivo activities against Plasmodium berghei (NT 173) are excellent.

In-vitro activity of pyronaridine and amodiaquine against African isolates (Senegal) of Plasmodium falciparum in comparison with standard antimalarial agents.

The in-vitro activities of pyronaridine, amodiaquine, chloroquine and quinine were evaluated against 161 isolates of Plasmodium falciparum from Senegal (Dielmo, Ndiop and Pikine), using an isotopic, micro, drug susceptibility test. The mean IC50 values (50% inhibitory concentration) for pyronaridine and amodiaquine were 3.8 nM (95% confidence interval (95% CI), 3.1-4.4) and 12.0 nM (95% CI, 10.0-14.0 nM), respectively. Pyronaridine and amodiaquine were more active than chloroquine against susceptible parasites. However, both drugs were significantly less active (P < 0.002 and P < 0.025) against chloroquine-resistant isolates than against chloroquine-susceptible isolates. Based on statistical calculation using the present data (mean IC50 + 2 S.D.), the cut-off value for in-vitro susceptibility to pyronaridine is IC50 < 15 nM; for eight isolates (5%) the IC50 was > 15 nM. No isolates tested showed resistance to amodiaquine (IC50 > 80 nM). Significant positive correlations, suggesting cross-resistance among these drugs in vitro, were found between pyronaridine and chloroquine (r2 = 0.19, P < 0.001), pyronaridine and quinine (r2 = 0.44, P < 0.001), pyronaridine and amodiaquine (r2 = 0.34, P < 0.001), amodiaquine and chloroquine (r2 = 0.14, P < 0.001), and amodiaquine and quinine (r2 = 0.21, P < 0.001). The present in-vitro findings require comparison with clinical studies.

In vitro activity of artemether against African isolates (Senegal) of Plasmodium falciparum in comparison with standard antimalarial drugs.

The in vitro activity of artemether against 56 African isolates of Plasmodium falciparum from Senegal was evaluated using an isotope-based drug susceptibility semi-microtest. The 50% inhibitory concentration (IC50) values for artemether were in a narrow range from 0.8 to 15.2 nM (mean IC50 = 3.43 nM) and the 95% confidence interval (CI) was 2.50-4.36 nM. Artemether was equally effective on chloroquine-sensitive and chloroquine-resistant isolates (mean IC50 = 346 nM, 95% CI = 2.08-4.84 nM versus mean IC50 = 2.80 nM, 95% CI = 2.00-3.60 nM). There was a significant positive correlation between responses to artemether and mefloquine (r2 = 0.36, P < 0.001), artemether and quinine (r2 = 0.085, P < 0.05), artemether and halofantrine (r2 = 0.075, P < 0.05), quinine and mefloquine (r2 = 0.205, P < 0.01), quinine and halofantrine (r2 = 0.124, P < 0.05), and mefloquine and halofantrine (r2 = 0.801, P < 0.001). A positive correlation between these drugs suggests in vitro cross-resistance or at least in vitro cross-susceptibility.

In vitro susceptibility of Gabonese wild isolates of Plasmodium falciparum to artemether, and comparison with chloroquine, quinine, halofantrine and amodiaquine.

The in vitro activity of artemether against 63 African isolates of Plasmodium falciparum from Libreville, Gabon was evaluated using an isotopic drug susceptibility semi-microtest. The 50% inhibitory concentration (IC50) values for artemether were in a narrow range from 0.8 to 34.8 nM (mean IC50 5.0 nM) and the 95% confidence interval (CI95%) was 3.6-6.3 nM. In vitro decreased susceptibility or resistance were observed with artemether (14%), to chloroquine (90%), to quinine (32%). Isolate susceptibility to amodiaquine and halofantrine was high i.e. 100% and 98%, respectively. There was a significant positive correlation between responses to artemether and amodiaquine (r2 = 0.45, P < 0.001), artemether and chloroquine (r2 = 0.36, P < 0.001), artemether and quinine (r2 = 0.31, P < 0.001), and artemether and halofantrine (r2 = 0.19, P < 0.01). Positive correlation between these drugs suggests in vitro cross-resistance or at least common features in drug uptake and/or mode of action or resistance.

Characterisation of the binding sites of monoclonal antibodies reacting with the Plasmodium falciparum rhoptry protein RhopH3.

Twenty one mouse monoclonal antibodies reacting or cross-reacting with the Plasmodium falciparum RhopH3 protein reacted with Ag44, a recombinant antigen expressing the 134 C-terminal RhopH3 residues. Using overlapping peptides scanning this region, two major binding sites were identified. The first one, recognised by eight anti-RhopH3 and seven cross-reacting mAbs, was mapped to the sequence Thr Asp Asn Thr Tyr or Thr Asp Asn Thr Tyr Lys (aa 823-828), depending on the support used for synthesis. Binding specificity and affinity were investigated for a subset of four mAbs reacting with this epitope, including one growth inhibitory mAb. Systematic replacements showed that the various mAbs had similar requirements. The inhibitory mAb presented a higher affinity for this sequence and bound to the adjacent sequence, Tyr Lys Glu Met Glu Leu (aa 827-832). A 2nd binding site, located around residue 850, was recognised by two anti-RhopH3 mAbs, which reacted exclusively with the 110 kDa RhopH3 polypeptide, unlike the other mAbs, which reacted with the 110 and 105 kDa RhopH3 antigens. This suggested that the 105 kDa RhopH3 polypeptide derives from the 110 kDa by C-terminal processing. Experimental evidence substantiating this conclusion was provided by the observation that antisera raised to peptides located upstream of the putative cleavage site reacted with both the 110 kDa and 105 kDa polypeptides, whereas antisera raised to the 45 C-terminal amino acids of RhopH3 reacted exclusively with the larger, 110 kDa product. The biological significance of this processing is discussed.

[Drug sensitivity of falciparum malaria imported into France in 1995]. / Chimiosensibilité du paludisme d'importation à Plasmodium falciparum en France en 1995.

The National Reference Centre for Malaria Chemosusceptibility (CNRCP) and the Tropical Medicine Institute of the Health Department for the Army (IMTSSA) monitor the chemosusceptibility of falciparum malaria introduced in France. In 1995, 353 isolates of P. falciparum are sent to the CNRCP and IMTSSA from malaria cases presenting in 49 civil and military hospitals distributed all over the french country. The patients are mostly Africans living in France and have mainly stayed in West Africa. Half of them did not take any chemoprophylaxis and a quarter took only chloroquine more or less regularly. The curative treatment, when known, is halofantrine alone in 52% of cases and quinine alone in 28% of cases. Three halofantrine failures are reported including 1 incorrect regimen and 4 quinine failures including 3 incorrect regimens. In 1995, in vitro resistance of P. falciparum isolates imported in France to the chemoprophylactic and therapeutic drugs is not worsening. In vitro quinine resistance is rare (1/108), mefloquine resistance (2/20) and halofantrine resistance (12/211) are limited, cycloguanil resistance (42/185) is stable and chloroquine resistance (84/230) is even decreasing (less selective pressure in Africa?).

[Malaria epidemic during a military-humanitarian mission in Africa]. / Epidémie de paludisme au cours d'une intervention militaro-humanitaire en Afrique.

A malaria epidemic broke out among French servicemen during a humanitarian military mission carried out in Central Africa in 1996. The purpose of this study was to determine compliance with drug prophylaxis for malaria by measuring blood levels of antimalarial drugs (combination treatment using chloroquine-proguanil or treatment with doxycycline) as well as to assess the conditions of vector control. The incidence density rate of malaria over a 60-day period was 3.1 cases per month per 100 men. Only reinforcement troops were affected. The risk of developing malaria was 5 times higher among new arrivals than in servicemen who had been in the zone for several months (95% CI relative risk = [2.9-7.8]). Type of prophylactic treatment had no effect on the incidence density rate. Study data showed that 40.2% of those treated for malaria were not in compliance with prophylactic treatment at the time of the malarial attack and that those who were in compliance with prophylaxis, i.e. the remaining 59.8%, presented a strain of plasmodium that was resistant to the prophylactic drugs at doses used. Findings also indicated the epidemic occurred mainly because operating conditions prevented implementation of proper vectorial control. The risk of epidemic could probably have been reduced by improving compliance with prophylactic treatment and changing standard vectorial control techniques, e.g. by using insecticide-treated uniforms.

Proguanil resistance in Plasmodium falciparum African isolates: assessment by mutation-specific polymerase chain reaction and in vitro susceptibility testing.

The antifolate proguanil is commonly used in the prophylaxis and treatment of Plasmodium falciparum malaria. A series of point mutations in the dihydrofolate reductase (DHFR) gene has been linked to differential susceptibility of varied P. falciparum clones or isolates to this drug. To survey the efficiency of proguanil prophylaxis in an African endemic region, and to evaluate the level of proguanil resistance in the corresponding parasite population, we performed drug susceptibility assays with P. falciparum isolates from Senegal, Kenya, and Niger. In parallel, we developed a mutation-specific polymerase chain reaction assay that enabled us to characterize mutations in the DHFR gene of the same isolates without in vitro parasite cultivation. We confirm previously available data showing that parasites harboring a point mutation from Ser108 to Asn present a decrease in susceptibility to cycloguanil (the active metabolite of proguanil), and we show that mutations in codons 51 and 59 appear to modulate the level of resistance to cycloguanil. No mutations in codons 16 and 164 were detected in resistant parasites, in contrast with results from some previous studies.

Electrocardiographic changes and halofantrine plasma level during acute falciparum malaria.

The aim of this study was 1) to assess the incidence of electrocardiographic changes after treatment with halofantrine and 2) to study the relationship between these changes and plasma levels of halofantrine and its main metabolite, N-desbutyl-halofantrine. Thirty-four male patients with uncomplicated falciparum malaria were enrolled in this study. Halofantrine was administered on two separate days at a total oral dosage of 24 mg/kg/day in three doses over a 12-hr period. The interval between the two treatments was seven days. Twelve-lead electrocardiography (ECG) was performed to measure the QT interval (QTc), ambulatory ECG monitoring was done to detect ventricular arrhythmia, signal-averaged ECG was performed to detect late ventricular potentials, and blood tests were performed to determine plasma concentrations of halofantrine and N-desbutyl-halofantrine. Maximum QTc was observed at 12 hr after both the first (P < 0.0002) and second treatments (P < 0.03). Signal-averaged ECG revealed late potentials in four cases (72 hr after the first treatment in one case and 24 hr after the second treatment in three cases). Ventricular arrhythmia was not observed. Significantly higher plasma concentrations of halofantrine were observed 2 hr after the second treatment. At this time, both the time effect and time interaction were significant (P < 0.008 and P < 0.02, respectively). The QTc interval was significantly correlated with the plasma halofantrine level (r = 0.41, P < 0.01) but not with the plasma N-desbutyl-halofantrine level (r = 0.30, not significant). In three cases, late ventricular potentials were associated with a maximum concentration of halofantrine. Our findings indicate that electrocardiographic changes are dose-dependent and that a second treatment at the same dosage may be hazardous.

[In vitro sensitivity of 85 Plasmodium falciparum isolates in the Fatick region, Senegal]. / Sensibilité in vitro de 85 isolats de Plasmodium falciparum dans la région de Fatick, Sénégal.

Increasing resistance of Plasmodium falciparum to chloroquine and other antimalarials has been reported in Africa. Only fragmentary data are available for Senegal. Although combined chloroquine-proguanil is soon scheduled for released on the market, few studies have been published concerning the susceptibility of isolates to either drug. The in vitro susceptibility of 85 Plasmodium falciparum isolates obtained between October 24 and December 2, 1995 from malaria-infected patients living in the Fatick Region of Senegal to five classical antimalarial drugs (chloroquine, cycloguanil, quinine, mefloquine, and halofantrine) was evaluated using an isotopic, semi-microtest. Twenty-nine percent of isolates were resistant to chloroquine (IC50 > 100 nM), 22% to cycloguanil (IC50 > 500 nM), 1% to quinine (IC50 > 500 nM), 22% to mefloquine (IC50 > 20 nM), and 8% to halofantrine (IC50 > 5 nM). Five percent of isolates were resistant to both chloroquine and cycloguanil. Positive correlation was observed between quinine and halofantrine activity and between mefloquine and halofantrine activity. These findings suggest cross resistance or reduced susceptibility between these antimalarial agents which all exhibit a methanolic function.

Human pharmacokinetics of chloroquine and proguanil delivered in a single capsule for malaria chemoprophylaxis.

Two antimalarial prophylactic regimens were compared in 17 healthy volunteers. Regimen A consisted of daily ingestion of a single capsule containing 100 mg base chloroquine (CQ) and 200 mg proguanil (PG). Regimen B consisted of daily ingestion of separate tablets of CQ (100 mg base) and PG (two 100 mg tablets). Both treatments lasted for 12 days. Effective chloroquine levels were reached after 72 hours with both regimens (49.9 ng/ml for treatment A and 36.7 ng/ml for treatment B). Proguanil and cycloguanil plasma levels were significantly lower on sampling obtained at H3 (three hours later) and H6 (six hours later) on day 1 in the regimen A (p < 0.002). Thereafter there were no significant difference between the two regimens. Both regimens were well tolerated, but regimen A using the capsule appeared better accepted and facilitates compliance.

Analysis of the high molecular weight rhoptry complex of Plasmodium falciparum using monoclonal antibodies.

Twenty-one monoclonal antibodies, obtained after immunization of mice with erythrocytic stages of Plasmodium falciparum, produced a double dot image in IFA. Immunoelectronmicroscopy indicated that the mAbs reacted with the rhoptries. Rhoptries are pear-shaped apical organelles, believed to be involved in invasion of the host cell by the parasite. The mAbs all immunoprecipitated the high molecular weight antigen complex. Some mAbs recognized on immunoblots only 1 protein of this complex, whereas others reacted with RhopH1 and RhopH3, or RhopH2 and RhopH3 or with the 3 proteins. An additional antigen of 52 kDa was also recognized by some of the mAbs. The epitopes defined by the mAbs were present in most of the 40 P. falciparum strains or isolates studied by IFA. Interestingly, the mAbs also reacted with high titres on P. vivax and P. ovale, but produced images that did not indicate an apical location. The mAbs failed to react on the non-human malaria parasites studied, P. cynomolgi and P. inui. On P. berghei or P. chabaudi parasites, only 5 mAbs gave a positive reaction, labelling a large network outside the parasite. Finally, the mAbs did not react with P. falciparum sporozoites, indicating that the rhoptries of merozoites and sporozoites, the two invasive stages of the malaria life-cycle are equipped with distinct sets of proteins.

Evolution of chloroquine resistance in central and west Africa.

The evolution of in vitro chloroquine susceptibility of clinical isolates of Plasmodium falciparum obtained from travellers returning to France was studied between 1986 and 1991 using the isotopic semi-microtest. Based on the analysis of 1,147 interpretable tests on isolates originating from Central and West Africa, the study showed that the proportion of chloroquine-resistant falciparum malaria remained stable between 1986 and 1988 and has diminished between 1989 and 1991. The diminution of chloroquine-resistant imported malaria may be associated, at least in part, with a better compliance of French travellers with the recommendation to use either mefloquine or a combination of chloroquine and proguanil since 1989 and an increasing proportion of African immigrants who tend to neglect regular chemoprophylaxis during the visit to their countries. The reason for the stabilisation of chloroquine resistance is unknown, and this phenomenon may be temporary, necessitating a continuous surveillance of drug susceptibility.