CQ-sensitive Strains Research Articles

Synthesis and Antimalarial Evaluation of New 1,3,5-tris[(4-(Substituted-aminomethyl)phenyl)methyl]benzene Derivatives: A Novel Alternative Antiparasitic Scaffold

A series of new 1,3,5-tris[(4-(substituted-aminomethyl)phenyl)methyl]benzene compounds were designed, synthesized, and evaluated in vitro against two parasites (Plasmodium falciparum and Leishmania donovani). The biological results showed antimalarial activity with IC50 values in the sub and μM range. The in vitro cytotoxicity of these new aza polyaromatic derivatives was also evaluated on human HepG2 cells. The 1,3,5-tris[(4-(substituted-aminomethyl)phenyl)methyl]benzene 1m was found as one of the most potent and promising antimalarial candidates with a ratio of cytotoxic to antiprotozoal activities of 83.67 against the P. falciparum CQ-sensitive strain 3D7. In addition, derivative 1r was also identified as the most interesting antimalarial compound with a selectivity index (SI) of 17.28 on the W2 P. falciparum CQ-resistant strain. It was previously described that the telomeres of P. falciparum could be considered as potential targets of these kinds of aza heterocycles; thus, the ability of these new derivatives to stabilize the parasitic telomeric G-quadruplexes was measured through a FRET melting assay.

Design, synthesis, and in silico-in vitro antimalarial evaluation of 1,2,3-triazole-linked dihydropyrimidinone quinoline hybrids

In response to the malaria parasite’s resistance towards quinoline-based antimalarial drugs, we have employed quinoline-containing compounds in combination with dihydropyrimidinone (DHPM) analogues as resistance reversal agents (RAs) and investigated their antimalarial activities based on DHPM’s resistance reversal abilities. The present study employed click chemistry to link DHPM and quinoline compounds which offered several synthetic advantages over the previously used amide coupling for the same hybrids. Among the synthesised compounds, 4 hybrids with the 7-chloroquinoline moiety showed antimalarial activity below 1 µM while compounds with the mefloquine moiety showed lower antimalarial activity than chloroquine (CQ) and the 7-chloroquinoline hybrids. Among the tested hybrids for the IC50 determination, four compounds displayed good antimalarial activity with increased sensitivity against the CQ-resistant K1 strain between 421 and 567 nM and showed higher activity between 138 and 245 nM against the NF54 CQ-sensitive strain, while three compounds have IC50 values greater than 5 µM. Additionally, in silico molecular docking and molecular dynamics studies were conducted to investigate the binding affinities of all the synthesised compounds as glutathione reductase protein competitive inhibitors. Further optimisation of the compound with the highest binding affinity generated 16 compounds with higher binding affinities than the flavine adenine dinucleotide (FAD) cofactor.

Enhancing malaria control using Lagenaria siceraria and its mediated zinc oxide nanoparticles against the vector Anopheles stephensi and its parasite Plasmodium falciparum

In many developing countries, there are certain health problems faced by the public, one among them is Malaria. This tropical disease is mainly caused by Plasmodium falciparum. It is categorized as a disaster to public health, which increases both mortality and morbidity. Numerous drugs are in practice to control this disease and their vectors. Eco-friendly control tools are required to battle against vector of this significant disease. Nanotechnology plays a major role in fighting against malaria. The present paper synthesized Zinc oxide nanoparticles (ZnO NPs) using zinc nitrate via simple green routes with the help of aqueous peel extract of Lagenaria siceraria (L. siceraria). The synthesized ZnO NPs were characterized by various biophysical methods. Moreover, the extract of L. siceraria and their mediated ZnO NPs was experimented against III instar larvae of An. stephensi. The impact of the treatment based on ZnO NPs concerning histology and morphology of mosquito larval was further observed. In the normal laboratory environment, the efficiency of predation of Poeciliareticulata (P. reticulata) against An. Stephensi larvae was found to be 44%, whereas in aqueous L. siceraria extract and its mediated ZnO NPs contaminated environment, P. reticulate showed predation efficiency of about 45.8% and 61.13% against An. Stephensi larva. L. siceraria synthesized ZnO NPs were examined against the Plasmodium falciparum CQ-sensitive strains. The L. siceraria extract and its mediated ZnO NPs showed the cytotoxic effects against HeLa cell lines with an IC50 value of 62.5 µg/mL. This study concludes that L. siceraria peel extract and L. siceraria synthesized ZnO NPs represent a valuable green option to fight against malarial vectors and parasites.

Design, synthesis, and antiprotozoal evaluation of new 2,4-bis[(substituted-aminomethyl)phenyl]quinoline, 1,3-bis[(substituted-aminomethyl)phenyl]isoquinoline and 2,4-bis[(substituted-aminomethyl)phenyl]quinazoline derivatives

A series of new 2,4-bis[(substituted-aminomethyl)phenyl]quinoline, 1,3-bis[(substituted-aminomethyl)phenyl]isoquinoline, and 2,4-bis[(substituted-aminomethyl)phenyl]quinazoline derivatives was designed, synthesised, and evaluated in vitro against three protozoan parasites (Plasmodium falciparum, Leishmania donovani, and Trypanosoma brucei brucei). Biological results showed antiprotozoal activity with IC50 values in the µM range. In addition, the in vitro cytotoxicity of these original molecules was assessed with human HepG2 cells. The quinoline 1c was identified as the most potent antimalarial candidate with a ratio of cytotoxic to antiparasitic activities of 97 against the P. falciparum CQ-sensitive strain 3D7. The quinazoline 3h was also identified as the most potent trypanosomal candidate with a selectivity index (SI) of 43 on T. brucei brucei strain. Moreover, as the telomeres of the parasites P. falciparum and Trypanosoma are possible targets of this kind of nitrogen heterocyclic compounds, we have also investigated stabilisation of the Plasmodium and Trypanosoma telomeric G-quadruplexes by our best compounds through FRET melting assays.

Uptake of a fluorescently tagged chloroquine analogue is reduced in CQ-resistant compared to CQ-sensitive Plasmodium falciparum parasites

BackgroundChloroquine (CQ) was the drug of choice for decades in the treatment of falciparum malaria until resistance emerged. CQ is suggested to accumulate in the parasite’s digestive vacuole (DV), where it unfolds its anti-malarial properties. Discrepancies of CQ accumulation in CQ-sensitive (CQS) and CQ-resistant (CQR) strains are thought to play a significant role in drug susceptibility. Analysis of CQ transport and intracellular localization using a fluorescently tagged CQ analogue could provide much needed information to distinguish susceptible from resistant parasite strains. The fluorescently tagged CQ analogue LynxTag-CQ™GREEN (CQGREEN) is commercially available and was assessed for its suitability.MethodsIC50 values were determined for both CQ and CQGREEN in two CQS and two CQR Plasmodium falciparum strains. Buffer solutions with varying pH were used to determine pH-dependent localization of CQGREEN in infected red blood cells. Before CQS or CQR parasites were exposed to different pH buffers, they were pre-loaded with varying concentrations of CQGREEN for up to 7 h. Intracellular accumulation was analysed using live cell confocal microscopy. CQGREEN uptake rates were determined for the cytosol and DV in the presence and absence of verapamil.ResultsIn CQS strains, twofold higher IC50 values were determined for the CQGREEN analogue compared to CQ. No significant differences in IC50 values were observed in CQR strains. Addition of verapamil reversed drug resistance of CQR strains to both CQ and CQGREEN. Live cell imaging revealed that CQGREEN fluorescence was mainly seen in the cytosol of most parasites, independent of the concentration used. Incubation periods of up to 7 h did not influence intracellular localization of CQGREEN. Nevertheless, CQGREEN uptake rates in CQR strains were reduced by 50% compared to CQS strains.ConclusionAlthough fluorescence of CQGREEN was mainly seen in the cytosol of parasites, IC50 assays showed comparable efficacy of CQGREEN and CQ in parasite killing of CQS and CQR strains. Reduced uptake rates of CQGREEN in CQR strains compared to CQS strains indicate parasite-specific responses to CQGREEN exposure. The data contains valuable information when CQGREEN is used as an analogue for CQ.

Synthesis, Antimalarial Evaluation and SAR Study of Some 1,3,5-Trisubstituted Pyrazoline Derivatives

The synthesis of a novel series of 1,3,5-trisubstitiuted pyrazoline was achieved by refluxing chalcone derivative with different heteroaryl hydrazines. The newly synthesized compounds were characterized by 1H NMR, 13CNMR, mass spectral and elemental analysis data. The synthetic series of novel pyrazoline hybrids was screened for in vitro schizont maturation assay against chloroquine sensitive 3D7 strain of Plasmodium falciparum. Most of the compounds showed promising in vitro antimalarial activity against CQ sensitive strain. The preliminary structure-activity relationship study showed that quinoline substituted analog at position N-1 showed maximum activity followed by benzothiazole substitution, while phenyl substitution lowers the antimalarial activity. The observed activity was persistent by the docking study on P. falciparum cystein protease falcipain-2. The pharmacokinetic properties were also studied using ADME prediction.

Primaquine and Chloroquine Fumardiamides as Promising Antiplasmodial Agents.

This paper describes a continuation of our efforts in the pursuit of novel antiplasmodial agents with optimized properties. Following our previous discovery of biologically potent asymmetric primaquine (PQ) and halogenaniline fumardiamides (1–6), we now report their significant in vitro activity against the hepatic stages of Plasmodium parasites. Furthermore, we successfully prepared chloroquine (CQ) analogue derivatives (11–16) and evaluated their activity against both the hepatic and erythrocytic stages of Plasmodium. Our results have shown that PQ fumardiamides (1–6) exert both higher activity against P. berghei hepatic stages and lower toxicity against human hepatoma cells than the parent drug and CQ derivatives (11–16). The favourable cytotoxicity profile of the most active compounds, 5 and 6, was corroborated by assays performed on human cells (human breast adenocarcinoma (MCF-7) and non-tumour embryonic kidney cells (HEK293T)), even when glucose-6-phosphate dehydrogenase (G6PD) was inhibited. The activity of CQ fumardiamides on P. falciparum erythrocytic stages was higher than that of PQ derivatives, comparable to CQ against CQ-resistant strain PfDd2, but lower than CQ when tested on the CQ-sensitive strain Pf3D7. In addition, both sets of compounds showed favourable drug-like properties. Hence, quinoline fumardiamides could serve as a starting point towards the development of safer and more effective antiplasmodial agents.

Synthesis, Biological Evaluation, and Molecular Modeling Studies of Chiral Chloroquine Analogues as Antimalarial Agents.

In a focused exploration, we designed, synthesized, and biologically evaluated chiral conjugated new chloroquine (CQ) analogues with substituted piperazines as antimalarial agents. In vitro as well as in vivo studies revealed that compound 7c showed potent activity (in vitro 50% inhibitory concentration, 56.98 nM for strain 3D7 and 97.76 nM for strain K1; selectivity index in vivo [up to at a dose of 12.5 mg/kg of body weight], 3,510) as a new lead antimalarial agent. Other compounds (compounds 6b, 6d, 7d, 7h, 8c, 8d, 9a, and 9c) also showed moderate activity against a CQ-sensitive strain (3D7) and superior activity against a CQ-resistant strain (K1) of Plasmodium falciparum Furthermore, we carried out docking and three-dimensional quantitative structure-activity relationship (3D-QSAR) studies of all in-house data sets (168 molecules) of chiral CQ analogues to explain the structure-activity relationships (SAR). Our new findings specify the significance of the H-bond interaction with the side chain of heme for biological activity. In addition, the 3D-QSAR study against the 3D7 strain indicated the favorable and unfavorable sites of CQ analogues for incorporating steric, hydrophobic, and electropositive groups to improve the antimalarial activity.

Synthesis and evaluation of anti-plasmodial and cytotoxic activities of epoxyazadiradione derivatives

Epoxyazadiradione (1), a major compound derived from Neem oil, showed modest anti-plasmodial activity against CQ-resistant and CQ-sensitive strains of the most virulent human malaria parasite P. falciparum. A series of analogues were synthesized by modification of the key structural moieties of this high yield natural product. Out of the library of all compounds tested, compounds 3c and 3g have showed modest anti-plasmodial activity against CQ-sensitive (IC50 2.8 ± 0.29 μM and 1.5 ± 0.01 μM) and CQ-resistant strains (IC50 1.3 ± 1.08 μM and 1.2 ± 0.14), while compounds 3k, 3l and 3m showed modest activity against CQ-sensitive strain of P. falciparum with IC50 values of 2.3 ± 0.4 μM, 2.9 ± 0.1 μM and 1.7 ± 0.06 μM, respectively. Additionally, cytotoxic properties of these derivatives against SIHA, PANC 1, MDA-MB-231, and IMR-3 cancer cell lines were also studied and the results indicated that low cytotoxic potentials of all the derivatives which indicating the high selectivity index of the compounds.

Antimalarial Efficacy of Thalictrum foliolosum (Meadow rue) Against Chloroquine-Resistant P. falciparum

Background: Investigation of traditionally used medicinal plants can provide safe and cost effective good antimalarials. The present study explores the in vitro antimalarial potential of Thalictrum foliolosum (meadow rue) leaf extract against Plasmodium falciparum. Methods: Phytochemical screening of the extract was carried out following standardized methods. WHO method based on schizont maturation inhibition was employed to assess the in vitro antiplasmodial activity of plant extract. Colorimetric MTT assay was used to evaluate the in vitro cytotoxicity of Thalictrum foliolosum. Selectivity Index was also calculated. Results: Phytochemical screening of the extract revealed presence of alkaloids, phenols, triterpenes, saponins and phytosterols. Ethanolic leaf extract of Thalictrum foliolosum (ELETF) exhibited IC50 1000μg/ml against both HeLa cells and normal fibroblasts. Selectivity index of ELETF was calculated to be >200 and =169.7 respectively for chloroquine resistant (RKL-9) and sensitive (MRC-2) strains of the parasite. Conclusions: Based on WHO recommendations ELETF can be classified as highly active antimalarial against CQ-resistant strain and possesses promising activity against CQ-sensitive strain. The high selectivity index (SI>10) also establishes Thalictrum foliolosum as an active antimalarial against both the strains of Plasmodium falciparum.

The Effect of Otostegia persica in Combination with Chloroquine on Chloroquine-Sensitive and Chloroquine-Resistant Strains of Plasmodium berghei Using In-vivo Fixed Ratios Method.

Malaria is one of the worldwide parasitic diseases which threaten the life of hundreds of millions of people at the malarious areas each year. The emergence of chloroquine-resistant strains of Plasmodium falciparum in most of the malarious areas has encountered the relevant countries with some difficulties about treating the acute cases of the disease particulary if the monotherapy regimen has been used. Because of many advantages for the combination therapy, the effectiveness of chloroquine (CQ) and Otostegia persica (OP), a medicinal plant in combination form, was tested against the chloroquine-sensitive and chloroquine-resistant strains of Plasmodium berghei in sourian mouse using in-vivo adapted fixed ratios method in this study. At the first step, ED50s (50% effective dose) of chloroquine and O. persica against both CQ-sensitive and CQ-resistant strains of P. berghei were calculated using in-vivo test in the mice. Ratios of 0, 10, 30, 50, 70, 90 and100% from each ED50 were prepared and contrarily combined together to make the following fixed ratios of 0/100, 10/90, 30/70, 50/50, 70/30, 90/10, and 100/0 of CQ/OP and the parasites were exposed to the combined ratios. Determination of ED50s showed 1.1 mg/Kg and 2.4 mg/Kg of mouse body weight for chloroquine in CQ-sensitive and CQ-resistant strains respectively and 450 mg/Kg for O. persica in both strains. The results also showed that the combinations of "50% CQ + 50% OP", "30% CQ + 70% O.P" and "70% CQ + 30% OP" were more effective than other combinations against CQ-sensitive strain. The fixed ratio combinations of chloroquine and O. persica showed an additive in CQ-resistant strain. Toxicity consideration showed no toxic effect of the combinations on the mice. Otostegia persica potentiated the effectiveness of chloroquine against the chloroquine-sensitive strain of P. berghei but not on chloroquine-resistant P. berghei. Moreover, the greatly modified fixed ratios method in this study can be considered as useful methods for in-vivo combination tests in murine malaria parasites.

Antimalarial activity of Carica papaya (Family: Caricaceae) leaf extract against Plasmodium falciparum

ObjectiveTo determine the antimalarial activity of the ethanol leaf extract of Carica papaya (C. papaya), blood stages of CQ-sensitive and CQ resistant strains against Plasmodium falciparum (P. falciparum) as target species. MethodsC. papaya leaf was collected in and around Kalveerampalyam village, Coimbatore, Tamil Nadu, India. C. papaya leaf was washed with tap water and shade dried at room temperature. An electrical blender powdered the dried plant materials (leaves). The powder 500g of the leaf was extracted with 1.5 L of organic solvents of ethanol for 8 h using a Soxhlet apparatus. The crude plant extracts were evaporated to dryness in rotary vacuum evaporator. One gram of the plant residue was dissolved in 100 mL of acetone (stock solution) and considered as 1% stock solution. From this stock solution, different concentrations were prepared ranging from 2%, 4%, 6%, 8% and 10%, respectively. ResultsThe highest larval mortality in the ethanol leaf extract of C. papaya against the 1st to 4th instars larvae and pupae values of LC50= 3.65%, 4.28%, 5.41%, 6.70%, and 7.50%, respectively. The LC90values of 9.61%, 11.75%, 13.53%, 16.36%, and 16.92%, respectively. Plant extracts showed moderate to good antiparasitic effects. These four concentrations (25, 50,100 and 150 μg/mL) of ethanol leaf extracts exhibited promising inhibitory activity against the CQ sensitive strain with (IC50) values 40.75%, 36.54%, 25.30%, and 18.0% and in CQ resistant 50.23%, 32.50%, 21.45%, and 23.12% against P. falciparum. ConclusionsIn conclusion, the results indicate the effective plant extracts have the potential to be used as an ideal eco-friendly approaches for the control of vector mosquitoes.

Identification of a Mutant PfCRT-Mediated Chloroquine Tolerance Phenotype in Plasmodium falciparum

Mutant forms of the Plasmodium falciparum transporter PfCRT constitute the key determinant of parasite resistance to chloroquine (CQ), the former first-line antimalarial, and are ubiquitous to infections that fail CQ treatment. However, treatment can often be successful in individuals harboring mutant pfcrt alleles, raising questions about the role of host immunity or pharmacokinetics vs. the parasite genetic background in contributing to treatment outcomes. To examine whether the parasite genetic background dictates the degree of mutant pfcrt-mediated CQ resistance, we replaced the wild type pfcrt allele in three CQ-sensitive strains with mutant pfcrt of the 7G8 allelic type prevalent in South America, the Oceanic region and India. Recombinant clones exhibited strain-dependent CQ responses that ranged from high-level resistance to an incremental shift that did not meet CQ resistance criteria. Nonetheless, even in the most susceptible clones, 7G8 mutant pfcrt enabled parasites to tolerate CQ pressure and recrudesce in vitro after treatment with high concentrations of CQ. 7G8 mutant pfcrt was found to significantly impact parasite responses to other antimalarials used in artemisinin-based combination therapies, in a strain-dependent manner. We also report clinical isolates from French Guiana that harbor mutant pfcrt, identical or related to the 7G8 haplotype, and manifest a CQ tolerance phenotype. One isolate, H209, harbored a novel PfCRT C350R mutation and demonstrated reduced quinine and artemisinin susceptibility. Our data: 1) suggest that high-level CQR is a complex biological process dependent on the presence of mutant pfcrt; 2) implicate a role for variant pfcrt alleles in modulating parasite susceptibility to other clinically important antimalarials; and 3) uncover the existence of a phenotype of CQ tolerance in some strains harboring mutant pfcrt.

Synthesis of oxalamide and triazine derivatives as a novel class of hybrid 4-aminoquinoline with potent antiplasmodial activity

Frequency of malaria and its resistance to chemotherapeutic options are emerging rapidly. To counter this problem, a series of 4-aminoquinolines having oxalamide and triazine functionalities in the side chain were synthesized and screened for their antimalarial activities. Triazine derivative 48 found to be the most active against CQ sensitive strain 3D7 of Plasmodium falciparum in an in vitro assay with an IC 50 of 5.23 ng/mL and oxalamide derivative 13 showed an in vivo suppression of 70.45% on day 4 against CQ resistant strain N-67 of Plasmodium yoelii.

Synthesis of novel thiourea, thiazolidinedione and thioparabanic acid derivatives of 4-aminoquinoline as potent antimalarials

In search of new 4-aminoquinolines which are not recognized by CQR mechanism, thiourea, thiazolidinedione and thioparabanic acid derivatives of 4-aminoquinoline were synthesized and screened for their antimalarial activities. Thiourea derivative 3 found to be the most active against CQ sensitive strain 3D7 of Plasmodium falciparum in an in vitro model with an IC 50 of 6.07 ng/mL and also showed an in vivo suppression of 99.27% on day 4 against CQ resistant strain N-67 of Plasmodium yoelii.

Synthesis and bioevaluation of hybrid 4-aminoquinoline triazines as a new class of antimalarial agents

The emergence and rapid spread of chloroquine resistant strains of Plasmodium falciparum has dramatically reduced the chemotherapeutic options. Towards this goal, a series of new class of hybrid 4-aminoquinoline triazines were synthesized and screened against CQ sensitive strain 3D7 of P. falciparum in an in vitro model. Compounds 65 and 69 exhibited more than 99% suppression on day 4 and on day 6 post treatment, compound 69 showed impressive 99.11% suppression against CQ resistant strain N-67 of P. yoelii in an in vivo assay.

16alpha-bromoepiandrosterone, a dehydroepiandrosterone (DHEA) analogue, inhibits Plasmodium falciparum and Plasmodium berghei growth.

Dehydroepiandrosterone (DHEA) and its analogue, 16alpha-bromoepiandrosterone (alpha-epi-Br), may have activity against viral and parasitic infections, including human immunodeficiency virus (HIV) and Cryptosporidium parvum. Therefore, we evaluated its antimalarial effects on Plasmodium falciparum and Plasmodium berghei. In vitro, chloroquine (CQ)-sensitive and resistant strains of P. falciparum parasitized red blood cells were incubated with escalating doses of alpha-epi-Br or CQ. In vivo, 62 rats were infected with P. berghei and treated with CQ or alpha-epi-Br. At the highest doses tested against a CQ-sensitive strain, parasitemias decreased from 25.4% in the saline control group to 4.3% and 4.8% in the alpha-epi-Br and CQ groups, respectively (P < 0.05). Against two CQ-resistant strains, parasitemias decreased from 22.3-28.8% and 24.8-30% in the CQ and saline groups, respectively, to 2.5-2.7% in the alpha-epi-Br groups (P = 0.003). In vivo, on Day 4, parasitemias decreased from 23% in the saline group to 9-12% and 12% in the in alpha-epi-Br and CQ groups, respectively (P < 0.05). These data demonstrate that alpha-epi-Br shows activity against CQ-sensitive and resistant strains of P. falciparum in vitro. At the doses tested against P. berghei in vivo in rats, alpha-epi-Br is comparable to CQ.

Evaluation of in vitro schizontocidal activity of plant parts of Calotropis procera—an ethnobotanical approach

Calotropis procera (Ait.) R.Br. commonly known, as ‘Arka’ is a popular medicinal plant found throughout the tropics of Asia and Africa and is used in many traditional systems of medicine. Important factors of the various parts of this plant have been widely reported. Good record keeping of subjective and objectively recorded cures by practitioners of traditional medicinal system will help in the establishment of the use of C. procera as an antimalarial plant. It has been attempted to see the effect of crude fractions of its flower, bud and roof against a chloroquine sensitive strain, MRC 20 and a chloroquine resistant strain, MRC 76 of Plasmodium falciparum using the Desjardins method and the effectiveness of its fractions compare better with the CQ sensitive strain than the CQ resistant strain in vitro.

Role for the Plasmodium falciparum Digestive Vacuole in Chloroquine Resistance

We have developed a method for the isolation of pure and intact Plasmodium falciparum digestive vacuoles capable of ATP-dependent chloroquine (CQ) accumulation in vitro. The method is rapid and reliable, and it produces a high yield of vacuoles (20%). CQ accumulation in isolated vacuoles was found to be ATP-, Mg 2+-, and temperature-dependent. We then investigated the CQ-accumulating capabilities of vacuoles isolated from CQ-resistant (CQR) and CQ-sensitive (CQS) parasites. At external CQ concentrations of 100 and 250 nM, vacuoles isolated from two CQS strains (D10 and RSA3) ( V max: 380–424 fmol/10 6 vacuoles/hr) accumulated significantly more CQ (∼3 times) than those isolated from three (FAC8, RSA11, and RSA15) of the four CQ-resistant strains of P. falciparum tested ( V max: 127–156 fmol/10 6 vacuoles/hr) ( P ≤ 0.05). We propose that the low level of CQ accumulation observed in vacuoles isolated from most of the CQ-resistant parasites tested contributes to the decreased CQ accumulation seen in these strains and, hence, to CQ resistance. Although it is often suggested that the digestive vacuole of the P. falciparum parasite is involved in the mechanism of CQ resistance, to our knowledge this is the first direct confirmation.