Treatment Failure in a UK Malaria Patient Harboring Genetically Variant Plasmodium falciparum From Uganda With Reduced In Vitro Susceptibility to Artemisinin and Lumefantrine.

BACKGROUND: Recent cases of clinical failure in malaria patients in the United Kingdom (UK) treated with artemether-lumefantrine have implications for malaria chemotherapy worldwide. METHODS: Parasites were isolated from an index case of confirmed Plasmodium falciparum treatment failure after standard treatment, and from comparable travel-acquired UK malaria cases. Drug susceptibility in vitro and genotypes at 6 resistance-associated loci were determined for all parasite isolates and compared with clinical outcomes for each parasite donor. RESULTS: A traveler, who returned to the UK from Uganda in 2022 with Plasmodium falciparum malaria, twice failed treatment with full courses of artemether-lumefantrine. Parasites from the patient exhibited significantly reduced susceptibility to artemisinin (ring-stage survival, 17.3% [95% confidence interval {CI}, 13.6%-21.1%]; P < .0001) and lumefantrine (effective concentration preventing 50% of growth = 259.4 nM [95% CI, 130.6-388.2 nM]; P = .001). Parasite genotyping identified an allele of pfk13 encoding both the A675V variant in the Pfk13 propeller domain and a novel L145V nonpropeller variant. In vitro susceptibility testing of 6 other P. falciparum lines of Ugandan origin identified reduced susceptibility to artemisinin and lumefantrine in 1 additional line, also from a 2022 treatment failure case. These parasites did not harbor a pfk13 propeller domain variant but rather the novel nonpropeller variant T349I. Variant alleles of pfubp1, pfap2mu, and pfcoronin were also identified among the 7 parasite lines. CONCLUSIONS: We confirm, in a documented case of artemether-lumefantrine treatment failure imported from Uganda, the presence of pfk13 mutations encoding L145V and A675V. Parasites with reduced susceptibility to both artemisinin and lumefantrine may be emerging in Uganda.

Measuring protective efficacy and quantifying the impact of drug resistance: A novel malaria chemoprevention trial design and methodology.

BACKGROUND: Recently revised WHO guidelines on malaria chemoprevention have opened the door to more tailored implementation. Countries face choices on whether to replace old drugs, target additional age groups, and adapt delivery schedules according to local drug resistance levels and malaria transmission patterns. Regular routine assessment of protective efficacy of chemoprevention is key. Here, we apply a novel modelling approach to aid the design and analysis of chemoprevention trials and generate measures of protection that can be applied across a range of transmission settings. METHODS AND FINDINGS: We developed a model of genotype-specific drug protection, which accounts for underlying risk of infection and circulating genotypes. Using a Bayesian framework, we fitted the model to multiple simulated scenarios to explore variations in study design, setting, and participant characteristics. We find that a placebo or control group with no drug protection is valuable but not always feasible. An alternative approach is a single-arm trial with an extended follow-up (>42 days), which allows measurement of the underlying infection risk after drug protection wanes, as long as transmission is relatively constant. We show that the currently recommended 28-day follow-up in a single-arm trial results in low precision of estimated 30-day chemoprevention efficacy and low power in determining genotype differences of 12 days in the duration of protection (power = 1.4%). Extending follow-up to 42 days increased precision and power (71.5%) in settings with constant transmission over this time period. However, in settings of unstable transmission, protective efficacy in a single-arm trial was overestimated by 24.3% if recruitment occurred during increasing transmission and underestimated by 15.8% when recruitment occurred during declining transmission. Protective efficacy was estimated with greater precision in high transmission settings, and power to detect differences by resistance genotype was lower in scenarios where the resistant genotype was either rare or too common. CONCLUSIONS: These findings have important implications for the current guidelines on chemoprevention efficacy studies and will be valuable for informing where these studies should be optimally placed. The results underscore the need for a comparator group in seasonal settings and provide evidence that the extension of follow-up in single-arm trials improves the accuracy of measures of protective efficacy in settings with more stable transmission. Extension of follow-up may pose logistical challenges to trial feasibility and associated costs. However, these studies may not need to be repeated multiple times, as the estimates of drug protection against different genotypes can be applied to different settings by adjusting for transmission intensity and frequency of resistance.

Baseline prevalence of molecular marker of sulfadoxine/pyrimethamine resistance in Ebonyi and Osun states, Nigeria: amplicon deep sequencing of dhps-540.

BACKGROUND: Chemoprevention plays an important role in malaria control strategy. Perennial malaria chemoprevention (PMC) using sulfadoxine/pyrimethamine (SP) is a WHO-approved strategy to combat malaria in young children and may lead to drug pressure. Introducing SP-PMC may therefore be compromised due to the emergence of Plasmodium falciparum resistant to SP, particularly mutation at K540E of the dihydropteroate synthase (dhps) gene. Molecular surveillance of resistance markers can support assessment of antimalarial efficacy and effectiveness. High prevalence of 540E is associated with reduced effectiveness of SP, and areas with more than 50% prevalence are considered unsuitable for intermittent preventative treatment in pregnancy (IPTp) implementation. Assessing 540E prevalence is an important undertaking before implementation of SP-PMC. METHODS: We conducted a rapid surveillance of dhps-540E to assess the suitability of SP as PMC in field studies from Ebonyi and Osun states in Nigeria. We used an in-house developed amplicon deep-sequencing method targeting part of the dhps gene. RESULTS: Our data reveal that 18.56% of individuals evaluated carried the 540E mutation mixed with the WT K540. Mutant variant 540E alone was not found, and 80% of isolates harboured only WT (K540). Clonal analysis of the sequencing data shows a very low proportion of 540E circulating in both states. CONCLUSIONS: Our data show that both states are suitable for SP-PMC implementation and, based on this finding, SP-PMC was implemented in Osun in 2022. Continuous monitoring of 540E will be required to ensure the chemoprevention effectiveness of SP in Nigeria.

The temporal dynamics of Plasmodium species infection after artemisinin-based combination therapy (ACT) among asymptomatic children in the Hohoe municipality, Ghana.

BACKGROUND: The routine surveillance of asymptomatic malaria using nucleic acid-based amplification tests is essential in obtaining reliable data that would inform malaria policy formulation and the implementation of appropriate control measures. METHODS: In this study, the prevalence rate and the dynamics of Plasmodium species among asymptomatic children (n = 1697) under 5 years from 30 communities within the Hohoe municipality in Ghana were determined. RESULTS AND DISCUSSION: The observed prevalence of Plasmodium parasite infection by polymerase chain reaction (PCR) was 33.6% (571/1697), which was significantly higher compared to that obtained by microscopy [26.6% (451/1697)] (P < 0.0001). Based on species-specific analysis by nested PCR, Plasmodium falciparum infection [33.6% (570/1697)] was dominant, with Plasmodium malariae, Plasmodium ovale and Plasmodium vivax infections accounting for 0.1% (1/1697), 0.0% (0/1697), and 0.0% (0/1697), respectively. The prevalence of P. falciparum infection among the 30 communities ranged from 0.0 to 82.5%. Following artesunate-amodiaquine (AS + AQ, 25 mg/kg) treatment of a sub-population of the participants (n = 184), there was a substantial reduction in Plasmodium parasite prevalence by 100% and 79.2% on day 7 based on microscopy and nested PCR analysis, respectively. However, there was an increase in parasite prevalence from day 14 to day 42, with a subsequent decline on day 70 by both microscopy and nested PCR. For parasite clearance rate analysis, we found a significant proportion of the participants harbouring residual Plasmodium parasites or parasite genomic DNA on day 1 [65.0% (13/20)], day 2 [65.0% (13/20)] and day 3 [60.0% (12/20)] after initiating treatment. Of note, gametocyte carriage among participants was low before and after treatment. CONCLUSION: Taken together, the results indicate that a significant number of individuals could harbour residual Plasmodium parasites or parasite genomic DNA after treatment. The study demonstrates the importance of routine surveillance of asymptomatic malaria using sensitive nucleic acid-based amplification techniques.

Genetic diversity of the Plasmodium falciparum GTP-cyclohydrolase 1, dihydrofolate reductase and dihydropteroate synthetase genes reveals new insights into sulfadoxine-pyrimethamine antimalarial drug resistance.

Plasmodium falciparum parasites resistant to antimalarial treatments have hindered malaria disease control. Sulfadoxine-pyrimethamine (SP) was used globally as a first-line treatment for malaria after wide-spread resistance to chloroquine emerged and, although replaced by artemisinin combinations, is currently used as intermittent preventive treatment of malaria in pregnancy and in young children as part of seasonal malaria chemoprophylaxis in sub-Saharan Africa. The emergence of SP-resistant parasites has been predominantly driven by cumulative build-up of mutations in the dihydrofolate reductase (pfdhfr) and dihydropteroate synthetase (pfdhps) genes, but additional amplifications in the folate pathway rate-limiting pfgch1 gene and promoter, have recently been described. However, the genetic make-up and prevalence of those amplifications is not fully understood. We analyse the whole genome sequence data of 4,134 P. falciparum isolates across 29 malaria endemic countries, and reveal that the pfgch1 gene and promoter amplifications have at least ten different forms, occurring collectively in 23% and 34% in Southeast Asian and African isolates, respectively. Amplifications are more likely to be present in isolates with a greater accumulation of pfdhfr and pfdhps substitutions (median of 1 additional mutations; P<0.00001), and there was evidence that the frequency of pfgch1 variants may be increasing in some African populations, presumably under the pressure of SP for chemoprophylaxis and anti-folate containing antibiotics used for the treatment of bacterial infections. The selection of P. falciparum with pfgch1 amplifications may enhance the fitness of parasites with pfdhfr and pfdhps substitutions, potentially threatening the efficacy of this regimen for prevention of malaria in vulnerable groups. Our work describes new pfgch1 amplifications that can be used to inform the surveillance of SP drug resistance, its prophylactic use, and future experimental work to understand functional mechanisms.

A molecular barcode to inform the geographical origin and transmission dynamics of Plasmodium vivax malaria.

Although Plasmodium vivax parasites are the predominant cause of malaria outside of sub-Saharan Africa, they not always prioritised by elimination programmes. P. vivax is resilient and poses challenges through its ability to re-emerge from dormancy in the human liver. With observed growing drug-resistance and the increasing reports of life-threatening infections, new tools to inform elimination efforts are needed. In order to halt transmission, we need to better understand the dynamics of transmission, the movement of parasites, and the reservoirs of infection in order to design targeted interventions. The use of molecular genetics and epidemiology for tracking and studying malaria parasite populations has been applied successfully in P. falciparum species and here we sought to develop a molecular genetic tool for P. vivax. By assembling the largest set of P. vivax whole genome sequences (n = 433) spanning 17 countries, and applying a machine learning approach, we created a 71 SNP barcode with high predictive ability to identify geographic origin (91.4%). Further, due to the inclusion of markers for within population variability, the barcode may also distinguish local transmission networks. By using P. vivax data from a low-transmission setting in Malaysia, we demonstrate the potential ability to infer outbreak events. By characterising the barcoding SNP genotypes in P. vivax DNA sourced from UK travellers (n = 132) to ten malaria endemic countries predominantly not used in the barcode construction, we correctly predicted the geographic region of infection origin. Overall, the 71 SNP barcode outperforms previously published genotyping methods and when rolled-out within new portable platforms, is likely to be an invaluable tool for informing targeted interventions towards elimination of this resilient human malaria.

The primate malaria parasites Plasmodium malariae, Plasmodium brasilianum and Plasmodium ovale spp.: genomic insights into distribution, dispersal and host transitions.

During the twentieth century, there was an explosion in understanding of the malaria parasites infecting humans and wild primates. This was built on three main data sources: from detailed descriptive morphology, from observational histories of induced infections in captive primates, syphilis patients, prison inmates and volunteers, and from clinical and epidemiological studies in the field. All three were wholly dependent on parasitological information from blood-film microscopy, and The Primate Malarias" by Coatney and colleagues (1971) provides an overview of this knowledge available at that time. Here, 50 years on, a perspective from the third decade of the twenty-first century is presented on two pairs of primate malaria parasite species. Included is a near-exhaustive summary of the recent and current geographical distribution for each of these four species, and of the underlying molecular and genomic evidence for each. The important role of host transitions in the radiation of Plasmodium spp. is discussed, as are any implications for the desired elimination of all malaria species in human populations. Two important questions are posed, requiring further work on these often ignored taxa. Is Plasmodium brasilianum, circulating among wild simian hosts in the Americas, a distinct species from Plasmodium malariae? Can new insights into the genomic differences between Plasmodium ovale curtisi and Plasmodium ovale wallikeri be linked to any important differences in parasite morphology, cell biology or clinical and epidemiological features?

The Role of Ultrasensitive Molecular Methods for Detecting Malaria-The Broader Perspective.

Ultrasensitive molecular diagnostics are lowering the limit of detection for malaria parasites in the blood and providing insights not captured by conventional tools such as microscopy and rapid antigen tests. Low-level malaria infections identified by molecular tools may influence clinical outcomes, transmission events, and elimination efforts. While many ultrasensitive molecular methods require well-equipped laboratories, technologies such as loop-mediated isothermal amplification and recombinase polymerase amplification provide more portable and analytically sensitive solutions. These tools may benefit asymptomatic patient screening, antenatal care, and elimination campaigns. We review the recent evidence, offer our perspective on the impact of these new tests, and identify future research priorities.

Persistent Submicroscopic Plasmodium falciparum Parasitemia 72 Hours after Treatment with Artemether-Lumefantrine Predicts 42-Day Treatment Failure in Mali and Burkina Faso.

A recent randomized controlled trial, the WANECAM (West African Network for Clinical Trials of Antimalarial Drugs) trial, conducted at seven centers in West Africa, found that artemether-lumefantrine, artesunate-amodiaquine, pyronaridine-artesunate, and dihydroartemisinin-piperaquine all displayed good efficacy. However, artemether-lumefantrine was associated with a shorter interval between clinical episodes than the other regimens. In a further comparison of these therapies, we identified cases of persisting submicroscopic parasitemia by quantitative PCR (qPCR) at 72 h posttreatment among WANECAM participants from 5 sites in Mali and Burkina Faso, and we compared treatment outcomes for this group to those with complete parasite clearance by 72 h. Among 552 evaluable patients, 17.7% had qPCR-detectable parasitemia at 72 h during their first treatment episode. This proportion varied among sites, reflecting differences in malaria transmission intensity, but did not differ among pooled drug treatment groups. However, patients who received artemether-lumefantrine and were qPCR positive at 72 h were significantly more likely to have microscopically detectable recurrent Plasmodium falciparum parasitemia by day 42 than those receiving other regimens and experienced, on average, a shorter interval before the next clinical episode. Haplotypes of pfcrt and pfmdr1 were also evaluated in persisting parasites. These data identify a possible threat to the parasitological efficacy of artemether-lumefantrine in West Africa, over a decade since it was first introduced on a large scale.

Plasmodium-associated changes in human odor attract mosquitoes.

Malaria parasites (Plasmodium) can change the attractiveness of their vertebrate hosts to Anopheles vectors, leading to a greater number of vector-host contacts and increased transmission. Indeed, naturally Plasmodium-infected children have been shown to attract more mosquitoes than parasite-free children. Here, we demonstrate Plasmodium-induced increases in the attractiveness of skin odor in Kenyan children and reveal quantitative differences in the production of specific odor components in infected vs. parasite-free individuals. We found the aldehydes heptanal, octanal, and nonanal to be produced in greater amounts by infected individuals and detected by mosquito antennae. In behavioral experiments, we demonstrated that these, and other, Plasmodium-induced aldehydes enhanced the attractiveness of a synthetic odor blend mimicking "healthy" human odor. Heptanal alone increased the attractiveness of "parasite-free" natural human odor. Should the increased production of these aldehydes by Plasmodium-infected humans lead to increased mosquito biting in a natural setting, this would likely affect the transmission of malaria.

The impact of delayed treatment of uncomplicated P. falciparum malaria on progression to severe malaria: A systematic review and a pooled multicentre individual-patient meta-analysis.

BACKGROUND: Delay in receiving treatment for uncomplicated malaria (UM) is often reported to increase the risk of developing severe malaria (SM), but access to treatment remains low in most high-burden areas. Understanding the contribution of treatment delay on progression to severe disease is critical to determine how quickly patients need to receive treatment and to quantify the impact of widely implemented treatment interventions, such as 'test-and-treat' policies administered by community health workers (CHWs). We conducted a pooled individual-participant meta-analysis to estimate the association between treatment delay and presenting with SM. METHODS AND FINDINGS: A search using Ovid MEDLINE and Embase was initially conducted to identify studies on severe Plasmodium falciparum malaria that included information on treatment delay, such as fever duration (inception to 22nd September 2017). Studies identified included 5 case-control and 8 other observational clinical studies of SM and UM cases. Risk of bias was assessed using the Newcastle-Ottawa scale, and all studies were ranked as 'Good', scoring ≥7/10. Individual-patient data (IPD) were pooled from 13 studies of 3,989 (94.1% aged <15 years) SM patients and 5,780 (79.6% aged <15 years) UM cases in Benin, Malaysia, Mozambique, Tanzania, The Gambia, Uganda, Yemen, and Zambia. Definitions of SM were standardised across studies to compare treatment delay in patients with UM and different SM phenotypes using age-adjusted mixed-effects regression. The odds of any SM phenotype were significantly higher in children with longer delays between initial symptoms and arrival at the health facility (odds ratio [OR] = 1.33, 95% CI: 1.07-1.64 for a delay of >24 hours versus ≤24 hours; p = 0.009). Reported illness duration was a strong predictor of presenting with severe malarial anaemia (SMA) in children, with an OR of 2.79 (95% CI:1.92-4.06; p < 0.001) for a delay of 2-3 days and 5.46 (95% CI: 3.49-8.53; p < 0.001) for a delay of >7 days, compared with receiving treatment within 24 hours from symptom onset. We estimate that 42.8% of childhood SMA cases and 48.5% of adult SMA cases in the study areas would have been averted if all individuals were able to access treatment within the first day of symptom onset, if the association is fully causal. In studies specifically recording onset of nonsevere symptoms, long treatment delay was moderately associated with other SM phenotypes (OR [95% CI] >3 to ≤4 days versus ≤24 hours: cerebral malaria [CM] = 2.42 [1.24-4.72], p = 0.01; respiratory distress syndrome [RDS] = 4.09 [1.70-9.82], p = 0.002). In addition to unmeasured confounding, which is commonly present in observational studies, a key limitation is that many severe cases and deaths occur outside healthcare facilities in endemic countries, where the effect of delayed or no treatment is difficult to quantify. CONCLUSIONS: Our results quantify the relationship between rapid access to treatment and reduced risk of severe disease, which was particularly strong for SMA. There was some evidence to suggest that progression to other severe phenotypes may also be prevented by prompt treatment, though the association was not as strong, which may be explained by potential selection bias, sample size issues, or a difference in underlying pathology. These findings may help assess the impact of interventions that improve access to treatment.

Novel Endochin-Like Quinolones Exhibit Potent In Vitro Activity against Plasmodium knowlesi but Do Not Synergize with Proguanil.

Quinolones, such as the antimalarial atovaquone, are inhibitors of the malarial mitochondrial cytochrome bc1 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. knowlesiin 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, <117 nM) and equally effective against P. falciparum We then screened selected quinolones and partner drugs using a longer exposure (2.5 life cycles) and found that proguanil is 10-fold less potent against P. knowlesi than P. falciparum, while the quinolones demonstrate similar potency. Finally, we used isobologram analysis to compare combinations of the ELQs with either proguanil or atovaquone. We show that all quinolone combinations with proguanil are synergistic against P. falciparum However, against P. knowlesi, no evidence of synergy between proguanil and the quinolones was found. Importantly, the combination of the novel quinolone ELQ-300 with atovaquone was synergistic against both species. Our data identify potentially important species differences in proguanil susceptibility and in the interaction of proguanil with quinolones and support the ongoing development of novel quinolones as potent antimalarials that target multiple species.

Plasmodium falciparum Isolates Carrying pfk13 Polymorphisms Harbor the SVMNT Allele of pfcrt in Northwestern Indonesia.

Artemisinin-based combination therapy (ACT) is the first-line antimalarial regimen in Indonesia. Susceptibility of Plasmodium falciparum to artemisinin is falling in the Greater Mekong subregion, but it is not known whether the efficacy of current combinations is also threatened in nearby Sumatera. We evaluated the genetic loci pfcrt, pfmdr1, and pfk13, considered to be under selection by artemisinin combination therapy, among 404 P. falciparum infections identified by PCR detection in a cross-sectional survey of 3,731 residents of three regencies. The pfcrt haplotype SVMNT (codons 72 to 76) was the most prevalent and displayed significant linkage disequilibrium with the pfmdr1 haplotype YY (codons 86 and 184) (odds ratio [OR] 26.7; 95% confidence interval [CI], 5.96 to 239.4; P < 0.001). This contrasts with Mekong countries, where the CVIET haplotype of pfcrt predominates. Among 231 evaluable isolates, only 9 (3.9%) showed any evidence of nonsynonymous gene variants in the propeller domain of pfk13 The Thr474Ala variant was seen in six individuals, and Cys580Tyr was identified with low confidence in only a single isolate from an asymptomatic individual. Among a subset of 117 symptomatic P. falciparum-infected individuals randomized to receive either dihydroartemisinin-piperaquine or artemether-lumefantrine, the treatment outcome was not associated with pretreatment genotype. However, submicroscopic persistent parasites at day 28 or day 42 of follow-up were significantly more likely to harbor the pfmdr1 haplotype NF (codons 86 and 184) than were pretreatment isolates (P < 0.001 for both treatment groups). Current ACT regimens appear to be effective in Sumatera, but evidence of persistent submicroscopic infection in some patients suggests further detailed studies of drug susceptibility should be undertaken.

An Individual Participant Data Population Pharmacokinetic Meta-analysis of Drug-Drug Interactions between Lumefantrine and Commonly Used Antiretroviral Treatment.

Treating malaria in HIV-coinfected individuals should consider potential drug-drug interactions. Artemether-lumefantrine is the most widely recommended treatment for uncomplicated malaria globally. Lumefantrine is metabolized by CYP3A4, an enzyme that commonly used antiretrovirals often induce or inhibit. A population pharmacokinetic meta-analysis was conducted using individual participant data from 10 studies with 6,100 lumefantrine concentrations from 793 nonpregnant adult participants (41% HIV-malaria-coinfected, 36% malaria-infected, 20% HIV-infected, and 3% healthy volunteers). Lumefantrine exposure increased 3.4-fold with coadministration of lopinavir-ritonavir-based antiretroviral therapy (ART), while it decreased by 47% with efavirenz-based ART and by 59% in the patients with rifampin-based antituberculosis treatment. Nevirapine- or dolutegravir-based ART and malaria or HIV infection were not associated with significant effects. Monte Carlo simulations showed that those on concomitant efavirenz or rifampin have 49% and 80% probability of day 7 concentrations <200 ng/ml, respectively, a threshold associated with an increased risk of treatment failure. The risk of achieving subtherapeutic concentrations increases with larger body weight. An extended 5-day and 6-day artemether-lumefantrine regimen is predicted to overcome these drug-drug interactions with efavirenz and rifampin, respectively.

Analysis of nuclear and organellar genomes of Plasmodium knowlesi in humans reveals ancient population structure and recent recombination among host-specific subpopulations.

The macaque parasite Plasmodium knowlesi is a significant concern in Malaysia where cases of human infection are increasing. Parasites infecting humans originate from genetically distinct subpopulations associated with the long-tailed (Macaca fascicularis (Mf)) or pig-tailed macaques (Macaca nemestrina (Mn)). We used a new high-quality reference genome to re-evaluate previously described subpopulations among human and macaque isolates from Malaysian-Borneo and Peninsular-Malaysia. Nuclear genomes were dimorphic, as expected, but new evidence of chromosomal-segment exchanges between subpopulations was found. A large segment on chromosome 8 originating from the Mn subpopulation and containing genes encoding proteins expressed in mosquito-borne parasite stages, was found in Mf genotypes. By contrast, non-recombining organelle genomes partitioned into 3 deeply branched lineages, unlinked with nuclear genomic dimorphism. Subpopulations which diverged in isolation have re-connected, possibly due to deforestation and disruption of wild macaque habitats. The resulting genomic mosaics reveal traits selected by host-vector-parasite interactions in a setting of ecological transition.

Modification of pfap2µ and pfubp1 Markedly Reduces Ring-Stage Susceptibility of Plasmodium falciparum to Artemisinin In Vitro.

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.

Geographical and temporal trends and seasonal relapse in Plasmodium ovale spp. and Plasmodium malariae infections imported to the UK between 1987 and 2015.

BACKGROUND: Plasmodium ovale spp. and P. malariae cause illness in endemic regions and returning travellers. Far less is known about these species than P. falciparum and P. vivax. METHODS: The UK national surveillance data, collected 1987 to 2015, were collated with the International Passenger Survey and climatic data to determine geographical, temporal and seasonal trends of imported P. ovale spp. and P. malariae infection. RESULTS: Of 52,242 notified cases of malaria, 6.04% (3157) were caused by P. ovale spp. and 1.61% (841) by P. malariae; mortality was 0.03% (1) and 0.12% (1), respectively. Almost all travellers acquired infection in West or East Africa. Infection rate per travel episode fell fivefold during the study period. The median latency of P. malariae and P. ovale spp. was 18 and 76 days, respectively; delayed presentation occurred with both species. The latency of P. ovale spp. infection imported from West Africa was significantly shorter in those arriving in the UK during the West African peak malarial season compared to those arriving outside it (44 days vs 94 days, p < 0.0001), implying that relapse synchronises with the period of high malarial transmission. This trend was not seen in P. ovale spp. imported from East Africa nor in P. malariae. CONCLUSION: In West Africa, where malaria transmission is highly seasonal, P. ovale spp. may have evolved to relapse during the malarial high transmission season. This has public health implications. Deaths are very rare, supporting current guidelines emphasising outpatient treatment. However, late presentations do occur.

Contribution of Plasmodium knowlesi to Multispecies Human Malaria Infections in North Sumatera, Indonesia.

Background: As Indonesia works toward the goal of malaria elimination, information is lacking on malaria epidemiology from some western provinces. As a basis for studies of antimalarial efficacy, we set out to survey parasite carriage in 3 communities in North Sumatera Province. Methods: A combination of active and passive detection of infection was carried out among communities in Batubara, Langkat, and South Nias regencies. Finger-prick blood samples from consenting individuals of all ages provided blood films for microscopic examination and blood spots on filter paper. Plasmodium species were identified using nested polymerase chain reaction (PCR) of ribosomal RNA genes and a novel assay that amplifies a conserved sequence specific for the sicavar gene family of Plasmodium knowlesi. Results: Of 3731 participants, 614 (16.5%) were positive for malaria parasites by microscopy. PCR detected parasite DNA in samples from 1169 individuals (31.3%). In total, 377 participants (11.8%) harbored P. knowlesi. Also present were Plasmodium vivax (14.3%), Plasmodium falciparum (10.5%) and Plasmodium malariae (3.4%). Conclusions: Amplification of sicavar is a specific and sensitive test for the presence of P. knowlesi DNA in humans. Subpatent and asymptomatic multispecies parasitemia is relatively common in North Sumatera, so PCR-based surveillance is required to support control and elimination activities.

pfk13-Independent Treatment Failure in Four Imported Cases of Plasmodium falciparum Malaria Treated with Artemether-Lumefantrine in the United Kingdom.

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.

Comparison of the susceptibility of Plasmodium knowlesi and Plasmodium falciparum to antimalarial agents.

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.