Search

Highlights • Malaria cases in UK and Ireland travellers can give false-negative HRP-RDT results. • Histidine-rich protein (HRP2/3) deletions can cause false-negative HRP-RDT results. • High parasitemia may give false-negative RDT results due to a prozone-like effect. • False-negative RDT results may also be a result of low parasite density. • Next-generation sequencing can elaborate the breakpoints of HRP2/3 deletions., Objectives Our objective was to systematically investigate false-negative histidine-rich protein 2 rapid diagnostic tests (HRP2-RDT) in imported Plasmodium falciparum malaria cases from travelers to the UK and the Republic of Ireland (RoI). Methods Five imported malaria cases in travellers returning to the UK and RoI from East Africa were reported to the PHE Malaria Reference Laboratory as negative according to histidine-rich protein (HRP2)-RDT. The cases were systematically investigated using microscopic, RDT, molecular, genomic, and in in vitro approaches. Results In each case, HRP2-RDT was negative, whereas microscopy confirmed the presence of P. falciparum. Further analysis revealed that the genes encoding HRP2 and HRP3 were deleted in three of the five cases. Whole-genome sequencing in one of these isolates confirmed deletions in P. falciparum chromosomes 8 and 13. Our study produced evidence that the fourth case, which had high parasitemia at clinical presentation, was a rare example of antigen saturation (‘prozone-like effect’), leading to a false negative in the HRP2-RDT, while the fifth case was due to low parasitemia. Conclusions False-negative HRP2-RDT results with P. falciparum are concerning. Our findings emphasise the necessity of supporting the interpretation of RDT results with microscopy, in conjunction with clinical observations, and sets out a systematic approach to identifying parasites carrying pfhrp2 and pfhrp3 deletions.

BackgroundPlasmodium knowlesi causes zoonotic malaria across Southeast Asia. First-line diagnostic microscopy cannot reliably differentiate P. knowlesi from other human malaria species. Rapid diagnostic tests (RDTs) designed for P. falciparum and P. vivax are used routinely in P. knowlesi co-endemic areas despite potential cross-reactivity for species-specific antibody targets.MethodsTen RDTs were evaluated: nine to detect clinical P. knowlesi infections from Malaysia, and nine assessing limit of detection (LoD) for P. knowlesi (PkA1-H.1) and P. falciparum (Pf3D7) cultures. Targets included Plasmodium-genus parasite lactate dehydrogenase (pan-pLDH) and P. vivax (Pv)-pLDH.ResultsSamples were collected prior to antimalarial treatment from 127 patients with microscopy-positive PCR-confirmed P. knowlesi mono-infections. Median parasitaemia was 788/µL (IQR 247-5,565/µL). Pan-pLDH sensitivities ranged from 50.6% (95% CI 39.6–61.5) (SD BIOLINE) to 87.0% (95% CI 75.1–94.6) (First Response® and CareStart™ PAN) compared to reference PCR. Pv-pLDH RDTs detected P. knowlesi with up to 92.0% (95% CI 84.3-96.7%) sensitivity (Biocredit™). For parasite counts ≥200/µL, pan-pLDH (Standard Q) and Pv-pLDH RDTs exceeded 95% sensitivity. Specificity of RDTs against 26 PCR-confirmed negative controls was 100%. Sensitivity of the 6 highest performing RDTs were not significantly different when comparing samples taken before and after (median 3 hours) antimalarial treatment. Parasite ring stages were present in 30% of pre-treatment samples, with ring stage proportions (mean 1.9%) demonstrating inverse correlation with test positivity of Biocredit™ and two CareStart™ RDTs.For cultured P. knowlesi, CareStart™ PAN demonstrated the lowest LoD at 25 parasites/µL; LoDs of other pan-pLDH ranged from 98 to >2000 parasites/µL. Pv-pLDH LoD for P. knowlesi was 49 parasites/µL. P. falciparum-pLDH or histidine-rich-protein-2 channels did not react with P. knowlesi.ConclusionSelected RDTs demonstrate sufficient performance for detection of all human malaria species including P. knowlesi in co-endemic areas where microscopy is not available, particularly for higher parasite counts, although cannot reliably differentiate among non-falciparum malaria.

The zoonotic parasite Plasmodium knowlesi has emerged as an important cause of human malaria in parts of Southeast Asia. The parasite is indistinguishable by microscopy from the more benign P. malariae, but can result in high parasitaemias with multiorgan failure, and deaths have been reported. Recognition of severe knowlesi malaria, and prompt initiation of effective therapy is therefore essential to prevent adverse outcomes. Here we review all studies reporting treatment of uncomplicated and severe knowlesi malaria. We report that although chloroquine is effective for the treatment of uncomplicated knowlesi malaria, artemisinin combination treatment is associated with faster parasite clearance times and lower rates of anaemia during follow-up, and should be considered the treatment of choice, particularly given the risk of administering chloroquine to drug-resistant P. vivax or P. falciparum misdiagnosed as P. knowlesi malaria in co-endemic areas. For severe knowlesi malaria, intravenous artesunate has been shown to be highly effective and associated with reduced case-fatality rates, and should be commenced without delay. Regular paracetamol may also be considered for patients with severe knowlesi malaria or for those with acute kidney injury, to attenuate the renal damage resulting from haemolysis-induced lipid peroxidation.

Several promising antimalarial drugs are currently being tested in human trials, such as artefenomel, cipargamin, ferroquine and ganaplacide. Many of these compounds were identified using high throughput screens against a single species of human malaria, Plasmodium falciparum, under the assumption that effectiveness against all malaria species will be similar, as has been observed for other antimalarial drugs. However, using our in vitro adapted line, we demonstrated recently that P. knowlesi is significantly less susceptible than P. falciparum to some new antimalarial drugs (e.g., cipargamin and DSM265), and more susceptible to others (e.g., ganaplacide). There is, therefore, an urgent need to determine the susceptibility profile of all human malaria species to the current generation of antimalarial compounds. We obtained ex vivo malaria samples from travellers returning to the United Kingdom and, using the [3H]hypoxanthine incorporation method, compared susceptibility to select established and experimental antimalarial agents among all major human infective Plasmodium species. We demonstrate that P. malariae and P. ovale spp. are significantly less susceptible than P. falciparum to cipargamin, DSM265 and AN13762, but are more susceptible to ganaplacide. Preliminary ex vivo data from single isolates of P. knowlesi and P. vivax demonstrate a similar profile. Our findings highlight the need to ensure cross species susceptibility profiles are determined early in the drug development pipeline. Our data can also be used to inform further drug development, and illustrate the utility of the P. knowlesi in vitro model as a scalable approach for predicting the drug susceptibility of non-falciparum malaria species in general., Graphical abstract Image 1, Highlights • P. knowlesi less susceptible than P. falciparum to some new antimalarials in vitro. • ex vivo malaria samples used to test susceptibility of other human malaria species. • P. malariae and P. ovale show similar drug susceptibility profile to P. knowlesi. • ex vivo drug screens from all malaria species can inform new drug development.

New antimalarial agents are identified and developed after extensive testing on Plasmodium falciparum parasites that can be grown in vitro. These susceptibility studies are important to inform lead optimisation and support further drug development. Until recently, little was known about the susceptibility of non-falciparum species as these had not been adapted to in vitro culture. The recent culture adaptation of P. knowlesi has therefore offered an opportunity to routinely define the drug susceptibility of this species, which is phylogenetically closer to all other human malarias than is P. falciparum. We compared the in vitro susceptibility of P. knowlesi and P. falciparum to a range of established and novel antimalarial agents under identical assay conditions. We demonstrated that P. knowlesi is significantly less susceptible than P. falciparum to six of the compounds tested; and notably these include three ATP4 inhibitors currently under development as novel antimalarial agents, and one investigational antimalarial, AN13762, which is 67 fold less effective against P. knowlesi. For the other compounds there was a less than two-fold difference in susceptibility between species. We then compared the susceptibility of a recent P. knowlesi isolate, UM01, to that of the well-established, older A1-H.1 clone. This recent isolate showed similar in vitro drug susceptibility to the A1-H.1 clone, supporting the ongoing use of the better characterised clone to further study drug susceptibility. Lastly, we used isobologram analysis to explore the interaction of a selection of drug combinations and showed similar drug interactions across species. The species differences in drug susceptibility reported by us here and previously, support adding in vitro drug screens against P. knowlesi to those using P. falciparum strains to inform new drug discovery and lead optimisation., Graphical abstract Image 1, Highlights • P. knowlesi >6-fold less susceptible to several ATP4 inhibitors than P. falciparum. • P. knowlesi equally susceptible to artemisinins and synthetic endoperoxides. • Isobolograms show similar drug interactions for P. knowlesi and P. falciparum. • New P. knowlesi isolate (UM01) similarly susceptible to antimalarials as A1-H.1

Background Many health facilities in malaria endemic countries are dependent on Rapid diagnostic tests (RDTs) for diagnosis and some National Health Service (NHS) hospitals without expert microscopists rely on them for diagnosis out of hours. The emergence of P. falciparum lacking the gene encoding histidine-rich protein 2 and 3 (HRP2 and HRP3) and escaping RDT detection threatens progress in malaria control and elimination. Currently, confirmation of RDT negative due to the deletion of pfhrp2 and pfhrp3, which encodes a cross-reactive protein isoform, requires a series of PCR assays. These tests have different limits of detection and many laboratories have reported difficulty in confirming the absence of the deletions with certainty. Methods We developed and validated a novel and rapid multiplex real time quantitative (qPCR) assay to detect pfhrp2, pfhrp3, confirmatory parasite and human reference genes simultaneously. We also applied the assay to detect pfhrp2 and pfhrp3 deletion in 462 field samples from different endemic countries and UK travellers. Results The qPCR assay demonstrated diagnostic sensitivity of 100% (n = 19, 95% CI= (82.3%; 100%)) and diagnostic specificity of 100% (n = 31; 95% CI= (88.8%; 100%)) in detecting pfhrp2 and pfhrp3 deletions. In addition, the assay estimates P. falciparum parasite density and accurately detects pfhrp2 and pfhrp3 deletions masked in polyclonal infections. We report pfhrp2 and pfhrp3 deletions in parasite isolates from Kenya, Tanzania and in UK travellers. Interpretation The new qPCR is easily scalable to routine surveillance studies in countries where P. falciparum parasites lacking pfhrp2 and pfhrp3 are a threat to malaria control.

Funder: Public Health England; doi: https://doi.org/10.13039/501100002141, Funder: European Developing Countries Trials Platform, Funder: Isaac Newton Trust, Funder: Alborada Fund, Funder: Global Health Innovative Technology Fund; doi: https://doi.org/10.13039/501100013996, Funder: Royal Society, New antimalarial therapeutics are needed to ensure that malaria cases continue to be driven down, as both emerging parasite resistance to frontline chemotherapies and mosquito resistance to current insecticides threaten control programmes. Plasmodium, the apicomplexan parasite responsible for malaria, causes disease pathology through repeated cycles of invasion and replication within host erythrocytes (the asexual cycle). Antimalarial drugs primarily target this cycle, seeking to reduce parasite burden within the host as fast as possible and to supress recrudescence for as long as possible. Intense phenotypic drug screening efforts have identified a number of promising new antimalarial molecules. Particularly important is the identification of compounds with new modes of action within the parasite to combat existing drug resistance and suitable for formulation of efficacious combination therapies. Here we detail the antimalarial properties of DDD01034957—a novel antimalarial molecule which is fast-acting and potent against drug resistant strains in vitro, shows activity in vivo, and possesses a resistance mechanism linked to the membrane transporter PfABCI3. These data support further medicinal chemistry lead-optimization of DDD01034957 as a novel antimalarial chemical class and provide new insights to further reduce in vivo metabolic clearance.

BackgroundRapid diagnostic tests (RDTs) that detect the malaria antigen histidine-rich protein 2 (HRP2) are widely used in endemic areas globally to confirm Plasmodium falciparum infection in febrile patients. The emergence of parasites lacking the gene encoding HRP2 and escaping RDT detection threatens progress in malaria control and elimination. Many health facilities in malaria endemic countries are dependent on RDTs for diagnosis and some National Health Service hospitals without expert microscopists rely on them for diagnosis out of hours. It is vital to study the emergence and the extent of such parasites globally to guide diagnostic policy. Currently, verification of the presence of such parasites in a blood sample requires a series of PCR assays to confirm the presence of P. falciparum and in the absence of amplicons from pfhrp2 and/or pfhrp3, which encodes a cross-reactive protein isoform. These tests have different limits of detection and many laboratories have reported difficulty in confirming the absence of pfhrp2 and pfhrp3 with certainty.MethodsWe developed and validated a novel and rapid multiplex real time quantitative (qPCR) assay to detect pfhrp2, pfhrp3, confirmatory parasite and human reference genes simultaneously. We also applied the assay to detect pfhrp2 and pfhrp3 deletion in 462 field samples from different endemic countries and UK travellers.ResultsThe qPCR assay showed limit of detection and quantification of 0.76-1.5 parasites per μl. The amplification efficiency, coefficient of determination (R2) and slope for the genes were 96-1.07%, 0.96-0.98 and −3.375 2 to −3.416 respectively. The assay demonstrated diagnostic sensitivity of 100% (n=19, 95% CI= (82.3%; 100%)) and diagnostic specificity of 100% (n=31; 95% CI= (88.8%; 100%)) in detecting pfhrp2 and pfhrp3 in. In addition, the qPCR assay estimates P. falciparum parasite density and can detect pfhrp2 and pfhrp3 deletions masked in polyclonal infections. We report pfhrp2 and pfhrp3 deletions in parasite isolates from Kenya, Tanzania and in UK travellers.ConclusionThe new qPCR assay is simple to use and offers significant advantages in speed and ease of interpretation. It is easily scalable to routine surveillance studies in countries where P. falciparum parasites lacking pfhrp2 and pfhrp3 are a threat to malaria control.

We examine in detail the AP-2 adaptin complex from the malaria parasite Plasmodium falciparum. In most studied organisms, AP-2 is involved in bringing material into the cell from outside, a process called endocytosis. Previous work shows that changes to the μ subunit of AP-2 can contribute to drug resistance. Our experiments show that AP-2 is essential for parasite development in blood but does not have any role in clathrin-mediated endocytosis. This suggests that a specialized function for AP-2 has developed in malaria parasites, and this may be important for understanding its impact on drug resistance., The efficacy of current antimalarial drugs is threatened by reduced susceptibility of Plasmodium falciparum to artemisinin, associated with mutations in pfkelch13. Another gene with variants known to modulate the response to artemisinin encodes the μ subunit of the AP-2 adaptin trafficking complex. To elucidate the cellular role of AP-2μ in P. falciparum, we performed a conditional gene knockout, which severely disrupted schizont organization and maturation, leading to mislocalization of key merozoite proteins. AP-2μ is thus essential for blood-stage replication. We generated transgenic P. falciparum parasites expressing hemagglutinin-tagged AP-2μ and examined cellular localization by fluorescence and electron microscopy. Together with mass spectrometry analysis of coimmunoprecipitating proteins, these studies identified AP-2μ-interacting partners, including other AP-2 subunits, the K10 kelch-domain protein, and PfEHD, an effector of endocytosis and lipid mobilization, but no evidence was found of interaction with clathrin, the expected coat protein for AP-2 vesicles. In reverse immunoprecipitation experiments with a clathrin nanobody, other heterotetrameric AP-complexes were shown to interact with clathrin, but AP-2 complex subunits were absent.

Quinolones, such as the antimalarial atovaquone, are inhibitors of the malarial mitochondrial cytochrome bc(1) complex, a target critical to the survival of both liver- and blood-stage parasites, making these drugs useful as both prophylaxis and treatment. Recently, several derivatives of endochin have been optimized to produce novel quinolones that are active in vitro and in animal models. While these quinolones exhibit potent ex vivo activity against Plasmodium falciparum and Plasmodium vivax, their activity against the zoonotic agent Plasmodium knowlesi is unknown. We screened several of these novel endochin-like quinolones (ELQs) for their activity against P. knowlesi in vitro and compared this with their activity against P. falciparum tested under identical conditions. We demonstrated that ELQs are potent against P. knowlesi (50% effective concentration

Management of uncomplicated malaria worldwide is threatened by the emergence in Asia of Plasmodium falciparum carrying variants of the pfk13 locus and exhibiting reduced susceptibility to artemisinin. Mutations in two other genes, ubp1 and ap2μ, are associated with artemisinin resistance in rodent malaria and with clinical failure of combination therapy in African malaria patients. Transgenic P. falciparum clones, each carrying orthologues of mutations in pfap2μ and pfubp1 associated with artemisinin resistance in Plasmodium chabaudi, were derived by Cas9 gene editing. Susceptibility to artemisinin and other antimalarial drugs was determined. Following exposure to 700 nM dihydroartemisinin in the ring-stage survival assay, we found strong evidence that transgenic parasites expressing the I592T variant (11% survival), but not the S160N variant (1% survival), of the AP2μ adaptin subunit were significantly less susceptible than the parental wild-type parasite population. The V3275F variant of UBP1, but not the V3306F variant, also displayed reduced susceptibility to dihydroartemisinin (8.5% survival versus 0.5% survival). AP2μ and UBP1 variants did not elicit reduced susceptibility to 48 h of exposure to artemisinin or to other antimalarial drugs. Therefore, variants of the AP2 adaptor complex μ-subunit and of the ubiquitin hydrolase UBP1 reduce in vitro artemisinin susceptibility at the early ring stage in P. falciparum These findings confirm the existence of multiple pathways to perturbation of either the mode of action of artemisinin, the parasite's adaptive mechanisms of resistance, or both. The cellular role of UBP1 and AP2μ in Plasmodium parasites should now be elucidated.

Background The simian malaria parasite Plasmodium knowlesi is now a well-recognized pathogen of humans in South-East Asia. Clinical infections appear adequately treated with existing drug regimens, but the evidence base for this practice remains weak. The availability of P. knowlesi cultures adapted to continuous propagation in human erythrocytes enables specific studies of in vitro susceptibility of the species to antimalarial agents, and could provide a surrogate system for testing investigational compounds against Plasmodium vivax and other non-Plasmodium falciparum infections that cannot currently be propagated in vitro. Objectives We sought to optimize protocols for in vitro susceptibility testing of P. knowlesi and to contrast outputs with those obtained for P. falciparum under comparable test conditions. Methods Growth monitoring of P. knowlesi in vitro was by DNA quantification using a SYBR Green fluorescent assay or by colorimetric detection of the lactate dehydrogenase enzyme. For comparison, P. falciparum was tested under conditions identical to those used for P. knowlesi. Results The SYBR Green I assay proved the most robust format over one (27 h) or two (54 h) P. knowlesi life cycles. Unexpectedly, P. knowlesi displays significantly greater susceptibility to the dihydrofolate reductase inhibitors pyrimethamine, cycloguanil and trimethoprim than does P. falciparum, but is less susceptible to the selective agents blasticidin and DSM1 used in parasite transfections. Inhibitors of dihydroorotate dehydrogenase also demonstrate lower activity against P. knowlesi. Conclusions The fluorescent assay system validated here identified species-specific P. knowlesi drug susceptibility profiles and can be used for testing investigational compounds for activity against non-P. falciparum malaria.

Management of uncomplicated malaria worldwide is threatened by the emergence in Asia of Plasmodium falciparum carrying variants of the pfk13 locus and exhibiting reduced susceptibility to artemisinin. Mutations in two other genes, ubp1 and ap2μ, are associated with artemisinin resistance in rodent malaria and with clinical failure of combination therapy in African malaria patients. Transgenic P. falciparum clones, each carrying orthologues of mutations in pfap2μ and pfubp1 associated with artemisinin resistance in Plasmodium chabaudi, were derived by Cas9 gene editing. Susceptibility to artemisinin and other antimalarial drugs was determined. Following exposure to 700 nM dihydroartemisinin in the ring-stage survival assay, we found strong evidence that transgenic parasites expressing the I592T variant (11% survival), but not the S160N variant (1% survival), of the AP2μ adaptin subunit were significantly less susceptible than the parental wild-type parasite population. The V3275F variant of UBP1, but not the V3306F variant, also displayed reduced susceptibility to dihydroartemisinin (8.5% survival versus 0.5% survival). AP2μ and UBP1 variants did not elicit reduced susceptibility to 48 h of exposure to artemisinin or to other antimalarial drugs. Therefore, variants of the AP2 adaptor complex μ-subunit and of the ubiquitin hydrolase UBP1 reduce in vitro artemisinin susceptibility at the early ring stage in P. falciparum. These findings confirm the existence of multiple pathways to perturbation of either the mode of action of artemisinin, the parasite’s adaptive mechanisms of resistance, or both. The cellular role of UBP1 and AP2μ in Plasmodium parasites should now be elucidated.

SummaryThe efficacy of current antimalarial drugs is threatened by reduced susceptibility of Plasmodium falciparum to artemisinin. In the Mekong region this is associated with mutations in the kelch propeller-encoding domain of pfkelch13, but variants of other parasite proteins are also thought to modulate the response to drug. Evidence from human and rodent studies suggests that the μ-subunit of the AP-2 adaptin trafficking complex is one such protein of interest. We generated transgenic Plasmodium falciparum parasites encoding the I592T variant of pfap2μ, orthologous to the I568T mutation associated with in vivo artemisinin resistance in P. chabaudi. When exposed to a four-hour pulse of dihydroartemisin in the ring-stage survival assay, two P. falciparum clones expressing AP-2μ I592T displayed significant and reproducible survival of 8.0% and 10.3%, respectively, compared to P. falciparum, K10. Conditional knockout indicates that the AP-2 trafficking complex in P. falciparum is essential for the fidelity of merozoite biogenesis and membrane organisation in the mature schizont. We also show that while other heterotetrameric AP-complexes and secretory factors interact with clathrin, AP-2 complex subunits do not. Thus, the AP-2 complex may be diverted from a clathrin-dependent endocytic role seen in most eukaryotes into a Plasmodium-specific function. These findings represent striking divergences from eukaryotic dogma and support a role for intracellular traffic in determining artemisinin sensitivity in vitro, confirming the existence of multiple functional routes to reduced ring-stage artemisinin susceptibility. Therefore, the utility of pfkelch13 variants as resistance markers is unlikely to be universal, and phenotypic surveillance of parasite susceptibility in vivo may be needed to identify threats to our current combination therapies.

Clinical studies suggest that outcomes for hospitalised malaria patients can be improved by managed hypothermia during treatment. We examined the impact of short pulses of low temperature on ring-stage susceptibility of Plasmodium falciparum to artemisinin in vitro. The usually artemisinin-sensitive clone 3D7 exhibited substantially reduced ring-stage susceptibility to a 4-h pulse of 700 nM dihydro-artemisinin administered during a 5-h pulse of low temperature down to 17 °C. Parasite growth through the subsequent asexual cycle was not affected by the temperature pulse. Chloroquine and pyronaridine susceptibility, in a standard 48-h test, was not affected by brief exposures to low temperature. Fever-like temperature pulses up to 40 °C were also accompanied by enhanced ring-stage survival of 700 nM artemisinin pulses, but parasite growth was generally attenuated at this temperature. We discuss these findings in relation to the possible activation of parasite stress responses, including the unfolded protein response, by hypo- or hyper-thermic conditions. Physiological states may need to be considered in artemisinin-treated P. falciparum patients., Graphical abstract Effect of transient hypothermia on ring-stage artemisinin susceptibility of P. falciparum clone 3D7. Ring-stage survival (proportion of control) of DHA pulse-treated 3D7 parasites (black circles) transiently incubated at hypothermic and normothermic temperatures. Open circles represent RSA survival estimates, in our hands, for previously described Cambodian parasite lines Cam 3.11 (reduced DHA susceptibility) and Cam 3.11REV (fully DHA susceptible), with K13 genotypes in superscript. P value was estimated by two-sided t-test between 17 °C (n = 4) and 37 °C (n = 6) (mean difference 58.7% survival; 95% CI 57.7–59.8% survival; 8 d.f.).Image 1

We demonstrate, for the first time, the multiplexed determination of microbial species from whole blood using the paper‐folding technique of origami to enable the sequential steps of DNA extraction, loop‐mediated isothermal amplification (LAMP), and array‐based fluorescence detection. A low‐cost handheld flashlight reveals the presence of the final DNA amplicon to the naked eye, providing a “sample‐to‐answer” diagnosis from a finger‐prick volume of human blood, within 45 min, with minimal user intervention. To demonstrate the method, we showed the identification of three species of Plasmodium, analyzing 80 patient samples benchmarked against the gold‐standard polymerase chain reaction (PCR) assay in an operator‐blinded study. We also show that the test retains its diagnostic accuracy when using stored or fixed reference samples.

This work was supported by Calgary Laboratory Services. Calgary Laboratory Services. Clinical Section of Microbiology. Calgary, Alberta, Canada / University of Calgary. Department of Microbiology, Immunology, and Infectious Diseases. Calgary, Alberta, Canada; Calgary Laboratory Services. Clinical Section of Microbiology. Calgary, Alberta, Canada Calgary Laboratory Services. Clinical Section of Microbiology. Calgary, Alberta, Canada McGill University. Department of Epidemiology, Biostatistics, and Occupational Health. Montreal, Quebec, Canada. University of Calgary. Department of Microbiology, Immunology, and Infectious Diseases. Calgary, Alberta, Canada / Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Laborat?rio de Pesquisas em Mal?ria. Ananindeua, PA, Brasil. ProvLab Alberta. Edmonton, Alberta, Canada. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Laborat?rio de Pesquisas em Mal?ria. Ananindeua, PA, Brasil. London School of Hygiene & Tropical Medicine. Faculty of Infectious & Tropical Diseases. Department of Immunology & Infection. London, UK. Calgary Laboratory Services. Clinical Section of Microbiology. Calgary, Alberta, Canada / University of Calgary, Calgary. Department Pathology and Laboratory Medicine. Alberta, Canada / University of Calgary. Department of Medicine. Calgary, Alberta, Canada. Calgary Laboratory Services. Clinical Section of Microbiology. Calgary, Alberta, Canada / University of Calgary. Department of Medicine. Calgary, Alberta, Canada. Calgary Laboratory Services. Clinical Section of Microbiology. Calgary, Alberta, Canada / University of Calgary. Department of Microbiology, Immunology, and Infectious Diseases. Calgary, Alberta, Canada / University of Calgary, Calgary. Department Pathology and Laboratory Medicine. Alberta, Canada / University of Calgary. Department of Medicine. Calgary, Alberta, Canada. The mainstay of malaria diagnosis relies on rapid diagnostic tests (RDTs) and microscopy, both of which lack analytical sensitivity. This leads to repeat testing to rule out malaria. A prospective diagnostic trial of the Meridian illumigene Malaria assay (loop-mediated isothermal amplification [LAMP]) was conducted comparing it with reference microscopy and RDTs (BinaxNOW Malaria) in returning travelers between June 2017 and January 2018. Returning travelers with signs and symptoms of malaria were enrolled in the study. RDTs, microscopy, and LAMP assays were performed simultaneously. A total of 298 patients (50.7% male; mean age, 32.5 years) were enrolled, most visiting friends and relatives (43.3%), presenting with fever (88.9%), not taking prophylaxis (82.9%), and treated as outpatients (84.1%). In the prospective arm (n = 348), LAMP had a sensitivity of 98.1% (95% confidence interval [CI], 90.0%-100%) and a specificity of 97.6% (95% CI, 95.2%-99.1%) vs microscopy. After discrepant resolution with real-time polymerase chain reaction, LAMP had a sensitivity of 100% (95% CI, 93.7%-100%) and a specificity of 100% (95% CI, 98.7%-100%) vs microscopy. After discrepant resolution, RDTs had a sensitivity of 83.3% (95% CI, 58.6%-96.4%) and a specificity of 96.2% (95% CI, 93.2%-98.1%) vs microscopy. When including retrospective specimens (n = 377), LAMP had a sensitivity of 98.8% (95% CI, 93.2%-100%) and a specificity of 97.6% (95% CI, 95.2%-99.1%) vs microscopy, and after discrepant resolution of this set, LAMP had a sensitivity of 100% (95% CI, 95.8%-100%) and a specificity of 100% (95% CI, 98.7%-100%). A cost-benefit analysis of reagents and labor suggests savings of up to USD$13 per specimen using a novel algorithm with LAMP screening.

Malaria, a disease caused by parasites of the genus Plasmodium, continues to claim hundreds of thousands of lives every year. Infusions of Cinchona bark provided the first effective cure for malaria in the early 1600s and were used widely for centuries. Quinine, isolated from the Cinchona bark in 1820, was to become the first identified antimalarial drug. Attempts to synthesise quinine chemically were unsuccessful but new synthetic antimalarials based on structural elements of quinine were pioneered by German chemists in the early twentieth century. From these compounds, chloroquine was to become one of the most successful antimalarial agents until drug resistance rendered it, and other subsequent antimalarials, ineffective. Several antimalarials have been developed in response to the growing resistance, the most important of which are the plant-derived artemisinin derivatives, discovered in China in the 1970s. Current antimalarial therapy recommends combinations of drugs over single-agent therapy to slow drug resistance. Key Concepts Malaria, a potentially lethal disease, is caused by parasites of the genus Plasmodium. Malarial infection affects hundreds of millions of individuals annually, chiefly within tropical regions of the world. From the seventeenth century, infusions of Cinchona bark were the only effective antimalarial treatment until its active component, quinine, was isolated in 1820. The first synthetic antimalarials originated from German drug discovery programmes in the 1920s and 1930s. The quinoline nucleus found in quinine became an important structural element of many synthetic antimalarials. Resistance to antimalarials continues to be a serious problem and drove much of the drug discovery in the twentieth century. Antimalarial combination therapies have replaced single-agent monotherapy in order to slow the development of drug resistance. Many antimalarials recommended for treatment today were first developed in China. Two of the most important antimalarials, quinine and artemisinin, are extracted from natural products (plants). Without a suitable vaccine, drugs remain important in treating malaria. Keywords: antimalarial agents; quinine; Cinchona bark; chloroquine; arteminisinin; antifolates

We present case histories of four patients treated with artemether-lumefantrine for falciparum malaria in UK hospitals in 2015 to 2016. Each subsequently presented with recurrent symptoms and Plasmodium falciparum parasitemia within 6 weeks of treatment with no intervening travel to countries where malaria is endemic. Parasite isolates, all of African origin, harbored variants at some candidate resistance loci. No evidence of pfk13 -mediated artemisinin resistance was found. Vigilance for signs of unsatisfactory antimalarial efficacy among imported cases of malaria is recommended.

To characterise plasmodial glycolysis, we generated two transgenic Plasmodium falciparum lines, one expressing P. falciparum hexokinase (PfHK) tagged with GFP (3D7-PfHK(GFP)) and another overexpressing native PfHK (3D7-PfHK(+)). Contrary to previous reports, we propose that PfHK is cytosolic. The glucose analogue, 2-deoxy-d-glucose (2-DG) was nearly 2-fold less toxic to 3D7-PfHK(+) compared with control parasites, supporting PfHK as a potential drug target. Although PfHK activity was higher in 3D7-PfHK(+), they accumulated phospho-[(14)C]2-DG at the same rate as control parasites. Transgenic parasites overexpressing the parasite's glucose transporter (PfHT) accumulated phospho-[(14)C]2-DG at a higher rate, consistent with glucose transport limiting glucose entry into glycolysis.

Poor quality antimalarials, including falsified, substandard, and degraded drugs, are a serious health concern in malaria-endemic countries. Guidelines are lacking on how to distinguish between substandard and degraded drugs. "Forced degradation" in an oven was carried out on three common artemisinin-based combination therapy (ACT) brands to detect products of degradation using liquid chromatography mass spectrometry and help facilitate classification of degraded drugs. "Natural aging" of 2,880 tablets each of ACTs artemether/lumefantrine and artesunate/amodiaquine was undertaken to evaluate their long-term stability in tropical climates. Samples were aged in the presence and absence of light on-site in Ghana and in a stability chamber (London), removed at regular intervals, and analyzed to determine loss of the active pharmaceutical ingredients (APIs) over time and detect products of degradation. Loss of APIs in naturally aged tablets (both in Ghana and the pharmaceutical stability chamber) was 0-7% over 3 years (∼12 months beyond expiry) with low levels of degradation products detected. Using this developed methodology, it was found that a quarter of ACTs purchased in Enugu, Nigeria (concurrent study), that would have been classified as substandard, were in fact degraded. Presence of degradation products together with evidence of insufficient APIs can be used to classify drugs as degraded.

Mutant forms of the Plasmodium falciparum chloroquine resistance transporter (PfCRT) mediate chloroquine resistance by effluxing the drug from the parasite's digestive vacuole, the acidic organelle in which chloroquine exerts its parasiticidal effect. However, different parasites bearing the same mutant form of PfCRT can vary substantially in their chloroquine susceptibility. Here, we have investigated the biochemical basis for the difference in chloroquine response among transfectant parasite lines having different genetic backgrounds but bearing the same mutant form of PfCRT. Despite showing significant differences in their chloroquine susceptibility, all lines with the mutant PfCRT showed a similar chloroquine-induced H + leak from the digestive vacuole, indicative of similar rates of PfCRT-mediated chloroquine efflux. Furthermore, all lines showed similarly reduced levels of drug accumulation. Factors other than chloroquine efflux and accumulation therefore influence the susceptibility to this drug in parasites expressing mutant PfCRT. Furthermore, in some but not all strains bearing mutant PfCRT, the 50% inhibitory concentration (IC 50 ) for chloroquine and the degree of resistance compared to that of recombinant control parasites varied with the length of the parasite growth assays. In these parasites, the 50% inhibitory concentration for chloroquine measured in 72- or 96-h assays was significantly lower than that measured in 48-h assays. This highlights the importance of considering the first- and second-cycle activities of chloroquine in future studies of parasite susceptibility to this drug.

The V-type H+ATPase is critical during the intraerythrocytic stage of the human malaria parasite Plasmodium falciparum. It is responsible for maintaining a near-neutral cytosolic pH (pH 7.3), an acidic digestive vacuole (pH 4.5-5.5) and the generation of an inside-negative plasma membrane potential (approximately -95 mV). Inhibition of this pump is therefore likely to result in profound physiological disturbances within the parasite and parasite death, as illustrated previously by the antiplasmodial activity of the potent and specific inhibitors of the V-type H+-ATPase, bafilomycin A(1) and concanamycin A. In this study we examined the antiplasmodial activity of a series of compounds previously designed, on the basis of the active structural constituents of bafilomycin A(1), to inhibit the osteoclast V-type H+-ATPase. The compounds were tested against up to 4 strains of P. falciparum with varying chloroquine sensitivities. Of the 30 novel compounds tested, 9 had sub-micromolar antiplasmodial IC(50) values, with the most active compound having an IC(50) of 160+/-20 nM. The activity of a number of these compounds was investigated in more detail. We show that these inhibitors acidify the parasite cytosol within seconds and that some inhibitors irreversibly kill the parasite within 0.5-4 h. The antiplasmodial activity of the V-type H+-ATPase inhibitors was strongly correlated with their ability to acidify the parasite cytosol (correlation coefficient 0.98). In combination studies, we show that the inhibitors act indifferently when combined with current antimalarials. Our data support the disruption of parasite pH regulation through inhibition of its V-type H+-ATPase as an antimalarial approach.

To survive, the human malaria parasite Plasmodium falciparum must acquire pantothenate (vitamin B5) from the external medium. Pantothenol (provitamin B5) inhibits parasite growth by competing with pantothenate for pantothenate kinase, the first enzyme in the coenzyme A biosynthesis pathway. In this study we investigated pantothenol uptake by P. falciparum and in doing so gained insights into the regulation of the parasite's coenzyme A biosynthesis pathway. Pantothenol was shown to enter P. falciparum-infected erythrocytes via two routes, the furosemide-inhibited "new permeation pathways" induced by the parasite in the infected erythrocyte membrane (the sole access route for pantothenate) and a second, furosemide-insensitive pathway. Having entered the erythrocyte, pantothenol is taken up by the intracellular parasite via a mechanism showing functional characteristics distinct from those of the parasite's pantothenate uptake mechanism. On reaching the parasite cytosol, pantothenol is phosphorylated and thereby trapped by pantothenate kinase, shown here to be under feedback inhibition control by coenzyme A. Furosemide reduced this inherent feedback inhibition by competing with coenzyme A for binding to pantothenate kinase, thereby increasing pantothenol uptake.

It is becoming increasingly apparent that certain mutations in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) alter the parasite's susceptibility to diverse compounds. Here we investigated the interaction of PfCRT with 3 tricyclic compounds that have been used to treat malaria (quinacrine [QC] and methylene blue [MB]) or to study P. falciparum (acridine orange [AO]). We measured the antiplasmodial activities of QC, MB, and AO against chloroquine-resistant and chloroquine-sensitive P. falciparum and determined whether QC and AO affect the accumulation and activity of chloroquine in these parasites. We also assessed the ability of mutant (PfCRT(Dd2)) and wild-type (PfCRT(D10)) variants of the protein to transport QC, MB, and AO when expressed at the surface of Xenopus laevis oocytes. Chloroquine resistance-conferring isoforms of PfCRT reduced the susceptibility of the parasite to QC, MB, and AO. In chloroquine-resistant (but not chloroquine-sensitive) parasites, AO and QC increased the parasite's accumulation of, and susceptibility to, chloroquine. All 3 compounds were shown to bind to PfCRT(Dd2), and the transport of QC and MB via this protein was saturable and inhibited by the chloroquine resistance-reverser verapamil. Our findings reveal that the PfCRT(Dd2)-mediated transport of tricyclic antimalarials reduces the parasite's susceptibility to these drugs.

Plasmodium falciparum , the most deadly agent of malaria, displays a wide variety of resistance mechanisms in the field. The ability of antimalarial compounds in development to overcome these must therefore be carefully evaluated to ensure uncompromised activity against real-life parasites. We report here on the selection and phenotypic as well as genotypic characterization of a panel of sensitive and multidrug-resistant P. falciparum strains that can be used to optimally identify and deconvolute the cross-resistance signals from an extended panel of investigational antimalarials. As a case study, the effectiveness of the selected panel of strains was demonstrated using the 1,2,4-oxadiazole series, a newly identified antimalarial series of compounds with in vitro activity against P. falciparum at nanomolar concentrations. This series of compounds was to be found inactive against several multidrug-resistant strains, and the deconvolution of this signal implicated pfcrt , the genetic determinant of chloroquine resistance. Targeted mode-of-action studies further suggested that this new chemical series might act as falcipain 2 inhibitors, substantiating the suggestion that these compounds have a site of action similar to that of chloroquine but a distinct mode of action. New antimalarials must overcome existing resistance and, ideally, prevent its de novo appearance. The panel of strains reported here, which includes recently collected as well as standard laboratory-adapted field isolates, is able to efficiently detect and precisely characterize cross-resistance and, as such, can contribute to the faster development of new, effective antimalarial drugs.

Resistance to quinoline antimalarials, especially to chloroquine and mefloquine has had a major impact on the treatment of malaria worldwide. In the period since 2000, significant progress has been made in understanding the origins of chloroquine resistance and to a lesser extent mefloquine resistance in Plasmodium falciparum. Chloroquine resistance correlates directly with mutations in the pfcrt gene of the parasite, while changes in another gene, pfmdr1, may also be related to chloroquine resistance in some strains. Mutations in pfcrt do not appear to correlate with mefloquine resistance, but some studies have implicated pfmdr1 in mefloquine resistance. Its involvement however, has not been definitively demonstrated. The protein products of these genes, PfCRT and Pgh-1 are both located in the food vacuole membrane of the parasite. Current evidence suggests that PfCRT is probably a transporter protein. Chloroquine appears to exit the food vacuole via this transporter in resistant PfCRT mutants. Pgh-1 on the other hand, resembles mammalian multi-drug resistance proteins and appears to be involved in expelling hydrophobic drugs from the food vacuole. Resistance reversing agents are believed to act by inhibiting these proteins. The currently known chloroquine- and mefloquine-resistance reversing agents are discussed in this review. This includes a discussion of structure-activity relationships in these compounds and hypotheses on their possible mechanisms of action. The status of current clinical applications is also briefly discussed.

Four new chemosensitisers against chloroquine-resistant Plasmodium falciparum based on the 9H-xanthene tricyclic scaffold were designed and synthesised in an attempt to identify simplified compounds that are easily accessible from commercially available starting materials. The compounds contain a common hydrophobic tricyclic 9H-xanthene moiety and an alkyl side chain with two amino groups, one of which is a tertiary substituted terminal amine, separated by three carbons and differing only in the chemical nature of the intermediary nitrogen atom. The best chemosensitising compound has a secondary amino group, showed a response modification index of 0.36 and caused a four-fold increase in chloroquine accumulation in a resistant strain of P. falciparum as well as having the highest selective therapeutic index when tested against a mammalian cell line.

Endocytosis is a fundamental process of eukaryotic cells and fulfills numerous functions, most notably, that of macromolecular nutrient uptake. Malaria parasites invade red blood cells and during their intracellular development endocytose large amounts of host cytoplasm for digestion in a specialized lysosomal compartment, the food vacuole. In the present study we have examined the effects of artemisinin and the quinoline drugs chloroquine and mefloquine on endocytosis in Plasmodium falciparum . By using novel assays we found that mefloquine and artemisinin inhibit endocytosis of macromolecular tracers by up to 85%, while the latter drug also leads to an accumulation of undigested hemoglobin in the parasite. During 5-h incubations, chloroquine inhibited hemoglobin digestion but had no other significant effect on the endocytic pathway of the parasite, as assessed by electron microscopy, the immunofluorescence localization of hemoglobin, and the distribution of fluorescent and biotinylated dextran tracers. By contrast, when chloroquine was added to late ring stage parasites, followed by a 12-h incubation, macromolecule endocytosis was inhibited by more than 40%. Moreover, there is an accumulation of transport vesicles in the parasite cytosol, possibly due to a disruption in vacuole-vesicle fusion. This fusion block is not observed with mefloquine, artemisinin, quinine, or primaquine but is mimicked by the vacuole alkalinizing agents ammonium chloride and monensin. These results are discussed in the light of present theories regarding the mechanisms of action of the antimalarials and highlight the potential use of drugs in manipulating and studying the endocytic pathway of malaria parasites.

Chemical analysis has shown that Plasmodium falciparum trophozoites contain 61±2% of the iron within parasitized erythrocytes, of which 92±6% is located within the food vacuole. Of this, 88±9% is in the form of haemozoin. 57Fe-Mössbauer spectroscopy shows that haemozoin is the only detectable iron species in trophozoites. Electron spectroscopic imaging confirms this conclusion.

Although efforts to understand the basis for inter-strain phenotypic variation in the most virulent malaria species, Plasmodium falciparum, have benefited from advances in genomic technologies, there have to date been few metabolomic studies of this parasite. Using 1H-NMR spectroscopy, we have compared the metabolite profiles of red blood cells infected with different P. falciparum strains. These included both chloroquine-sensitive and chloroquine-resistant strains, as well as transfectant lines engineered to express different isoforms of the chloroquine-resistance-conferring pfcrt (P. falciparum chloroquine resistance transporter). Our analyses revealed strain-specific differences in a range of metabolites. There was marked variation in the levels of the membrane precursors choline and phosphocholine, with some strains having >30-fold higher choline levels and >5-fold higher phosphocholine levels than others. Chloroquine-resistant strains showed elevated levels of a number of amino acids relative to chloroquine-sensitive strains, including an approximately 2-fold increase in aspartate levels. The elevation in amino acid levels was attributable to mutations in pfcrt. Pfcrt-linked differences in amino acid abundance were confirmed using alternate extraction and detection (HPLC) methods. Mutations acquired to withstand chloroquine exposure therefore give rise to significant biochemical alterations in the parasite., The metabolite profiles of red blood cells infected with different malaria parasite strains were compared. Amino acid profiles varied with the chloroquine resistance status of the strain, and this was linked specifically to mutations in the parasite's chloroquine resistance transporter.

The emergence of drug-resistant parasites is a serious threat faced by malaria control programs. Understanding the genetic basis of resistance is critical to the success of treatment and intervention strategies. A novel locus associated with antimalarial resistance, ap2-mu (encoding the mu chain of the adaptor protein 2 [AP2] complex), was recently identified in studies on the rodent malaria parasite Plasmodium chabaudi (pcap2-mu). Furthermore, analysis in Kenyan malaria patients of polymorphisms in the Plasmodium falciparum ap2-mu homologue, pfap2-mu, found evidence that differences in the amino acid encoded by codon 160 are associated with enhanced parasite survival in vivo following combination treatments which included artemisinin derivatives. Here, we characterize the role of pfap2-mu in mediating the in vitro antimalarial drug response of P. falciparum by generating transgenic parasites constitutively expressing codon 160 encoding either the wild-type Ser (Ser160) or the Asn mutant (160Asn) form of pfap2-mu. Transgenic parasites carrying the pfap2-mu 160Asn allele were significantly less sensitive to dihydroartemisinin using a standard 48-h in vitro test, providing direct evidence of an altered parasite response to artemisinin. Our data also provide evidence that pfap2-mu variants can modulate parasite sensitivity to quinine. No evidence was found that pfap2-mu variants contribute to the slow-clearance phenotype exhibited by P. falciparum in Cambodian patients treated with artesunate monotherapy. These findings provide compelling evidence that pfap2-mu can modulate P. falciparum responses to multiple drugs. We propose that this gene should be evaluated further as a potential molecular marker of antimalarial resistance.

Contains fulltext : 139083.pdf (Publisher’s version ) (Open Access) BACKGROUND: The efficacy of artemisinin-based combination therapy (ACT) for Plasmodium falciparum malaria may be threatened by parasites with reduced responsiveness to artemisinins. Among 298 ACT-treated children from Mbita, Kenya, submicroscopic persistence of P. falciparum on day 3 posttreatment was associated with subsequent microscopically detected parasitemia at days 28 or 42. METHODS: DNA sequences of resistance-associated parasite loci pfcrt, pfmdr1, pfubp1, and pfap2mu were determined in the Mbita cohort before treatment, on days 2 and 3 after initiation of treatment, and on the day of treatment failure. RESULTS: Parasites surviving ACT on day 2 or day 3 posttreatment were significantly more likely than the baseline population to carry the wild-type haplotypes of pfcrt (CVMNK at codons 72-76; P < .001) and pfmdr1 (NFD at codons 86, 184, 1246; P < .001). In contrast, variant alleles of the novel candidate resistance genes pfap2mu (S160N/T; P = .006) and pfubp-1 (E1528D; P < .001) were significantly more prevalent posttreatment. No genetic similarities were found to artemisinin-tolerant parasites recently described in Cambodia. CONCLUSIONS: Among treated children in western Kenya, certain P. falciparum genotypes defined at pfcrt, pfmdr1, pfap2mu, and pfubp1 more often survive ACT at the submicroscopic level, and contribute to onward transmission and subsequent patent recrudescence.

Research into chloroquine resistance reversal in Plasmodium falciparum has revealed a widespread range of functionally and structurally diverse chemosensitizers. However, nearly all of these chemosensitizers reverse resistance optimally only at concentrations that are toxic to humans. Verapamil, desipramine, and trifluoperazine were shown to potentiate chloroquine accumulation in a chloroquine-resistant (CQ r ) strain of P. falciparum, while progesterone, ivermectin, and cyclosporin A were not shown to potentiate chloroquine accumulation. The simultaneous use of two or even three of these chemosensitizers at concentrations within their therapeutic ranges in humans displayed an additive effect in potentiating chloroquine accumulation in the CQ r strain. The levels of resistance reversal achieved with these multiple combinations were comparable to those achieved with high concentrations of the single agents used to enhance the activity of chloroquine. No chemosensitizer, whether used singly or in combination, potentiated any change in chloroquine accumulation or a shift in the 50% inhibitory concentration for the chloroquine-sensitive strain. The use of combinations of chemosensitizers at concentrations not toxic to humans could effectively reverse chloroquine resistance without the marked toxicity from the use of a single agent at high concentrations. This cocktail of chemosensitizers may serve as a viable treatment to restore the efficacy of chloroquine in patients with malaria. The spread of chloroquine (CQ) resistance in Plasmodium falciparum throughout most areas where malaria is endemic has necessitated alternate treatments for malaria. More recently, antimalarials such as mefloquine and halofantrine were developed, but indications are that these are becoming ineffective as resistance to them spreads (20). There have been attempts to restore CQ’s efficacy in vitro and in vivo by using it in combination with resistance reversers like promethazine and chlorpheniramine (16, 18). However, these compounds, which stimulate the uptake of CQ by resistant strains and drastically reduce the 50% inhibitory concentration (IC50), operate optimally as resistance reversers in vitro only at concentrations that are highly toxic in vivo. Work with multidrug-resistant (MDR) cancer cells has shown that it is possible to reverse anticancer agent resistance by using combinations of chemosensitizers at concentrations not toxic to humans (10). The levels of reversal obtained with these combinations were comparable to those obtained with the single agents used at their optimal concentrations. In P. falciparum, two calcium channel blockers, verapamil (VPL) and fantofarone, have been shown to act synergistically in reversing CQ resistance (1). We selected several structurally and functionally diverse compounds to test CQ resistance (CQ r ) reversal in P. falciparum. VPL and desipramine (DES) are known resistance reversers in P. falciparum (2, 14). However, progesterone (PROG), ivermectin (IVM), trifluoperazine (TRF), and cyclosporine (CsA) have not been implicated in CQ resistance reversal, although they do reverse multidrug resistance in cancer cells (7, 11, 17). A combination of the chemosensitizers used at low concentrations was shown to work as effectively in vitro in reversing CQ resistance as the single compounds used at their optimal concentrations with CQ. This may yet prove to be an effective way of overcoming the CQ resistance without the toxicity associated with these chemosensitizers in vivo.

Amine and urea analogs of ferrochloroquine with varying methylene spacer lengths were synthesised, studied by cyclic voltammetry and evaluated in vitro against a sensitive (D10) and resistant (K1) strain of Plasmodium falciparum. Most analogs were found to be more active than chloroquine in both strains. In D10 ureas were more active than amines and antimalarial activity in this strain correlated well with the length of the methylene spacer and redox potentials. The length of the methylene spacer was a major determinant of antimalarial activity in K1.

The intraerythrocytic malaria parasite is susceptible to oxidative stress and this may play a role in the mechanism of action of some antimalarial agents. Here we show that exposure of the intraerythrocytic malaria parasite to the oxidising agent hydrogen peroxide results in a fall in the intracellular ATP level and inhibition of the parasite's V-type H(+)-ATPase, causing a loss of pH control in both the parasite cytosol and the internal digestive vacuole. In contrast to the V-type H(+)-ATPase, the parasite's digestive vacuole H(+)-pyrophosphatase is insensitive to hydrogen peroxide-induced oxidative stress. This work provides insights into the effects of oxidative stress on the intraerythrocytic parasite, as well as providing an alternative possible explanation for a previous report that light-induced oxidative stress causes selective lysis of the parasite's digestive vacuole.

The intraerythrocytic stage of the human malaria parasite Plasmodium falciparum relies on glycolysis for ATP generation, and because it has no energy stores, a constant supply of glucose is necessary for the parasite to grow and multiply. The 2-substituted glucose analogs 2-deoxy-D-glucose (2-DG) and 2-fluoro-2-deoxy-D-glucose (2-FG) have been previously shown to inhibit the in vitro growth of P. falciparum and have been suggested to do so by inhibiting glycosylation in the parasite. In this study, we have investigated the antiplasmodial mechanism of action of 2-DG and 2-FG and compared it with that of other 2-substituted-glucose analogs. The compounds tested inhibited parasite growth to varying degrees, with 2-FG being the most effective. The antiplasmodial activity of some, but not all, of the analogs could be altered by varying the glucose concentration in the culture medium, increasing the antiplasmodial activity of the analogs as the glucose concentration is reduced. A trend was observed between the antiplasmodial activity of these analogs and their ability to inhibit glucose accumulation, glucose phosphorylation by hexokinase, and cytosolic pH regulation within the intraerythrocytic stage of the parasite. Our data are consistent with inhibition of glycolysis being a primary mechanism by which 2-DG and 2-FG inhibit parasite growth, and they validate the early steps in glycolysis as viable drug targets.