Delayed Parasite Clearance Research Articles

Evolution and Genetic Diversity of the k13 Gene Associated with Artemisinin Delayed Parasite Clearance in Plasmodium falciparum.

Mutations in the Plasmodium falciparumk13 (Pfk13) gene are linked to delayed parasite clearance in response to artemisinin-based combination therapies (ACTs) in Southeast Asia. To explore the evolutionary rate and constraints acting on this gene, k13 orthologs from species sharing a recent common ancestor with P. falciparum and Plasmodium vivax were analyzed. These comparative studies were followed by genetic polymorphism analyses within P. falciparum using 982 complete Pfk13 sequences from public databases and new data obtained by next-generation sequencing from African and Haitian isolates. Although k13 orthologs evolve at heterogeneous rates, the gene was conserved across the genus, with only synonymous substitutions being found at residues where mutations linked to the delayed parasite clearance phenotype have been reported. This suggests that those residues were under constraint from undergoing nonsynonymous changes during evolution of the genus. No fixed nonsynonymous differences were found between Pfk13 and its orthologs in closely related species found in African apes. This indicates that all nonsynonymous substitutions currently found in Pfk13 are younger than the time of divergence between P. falciparum and its closely related species. At the population level, no mutations linked to delayed parasite clearance were found in our samples from Africa and Haiti. However, there is a high number of single Pfk13 mutations segregating in P. falciparum populations, and two predominant alleles are distributed worldwide. This pattern is discussed in terms of how changes in the efficacy of natural selection, affected by population expansion, may have allowed for the emergence of mutations tolerant to ACTs.

Clinical impact of the two ART resistance markers, K13 gene mutations and DPC3 in Vietnam.

BackgroundIn Vietnam, a rapid decline of P. falciparum malaria cases has been documented in the past years, the number of Plasmodium falciparum malaria cases has rapidly decreased passing from 19.638 in 2012 to 4.073 cases in 2016. Concomitantly, the spread of artemisinin resistance markers is raising concern on the future efficacy of the ACTs. An evaluation of the clinical impact of the artemisinin resistance markers is therefore of interest.MethodsThe clinical effectiveness of dihydroartemisinin-piperaquine therapy (DHA-PPQ) has been evaluated in three districts characterized by different rates of ART resistance markers: K13(C580Y) mutation and delayed parasite clearance on day 3 (DPC3). Patients were stratified in 3 groups a) no markers, b) one marker (suspected resistance), c) co-presence of both markers (confirmed resistance). In the studied areas, the clinical effectiveness of DHA-PPQ has been estimated as malaria recrudescence within 60 days.ResultsThe rate of K13(C580Y) ranged from 75.8% in Krong Pa to 1.2% in Huong Hoa district. DPC3 prevalence was higher in Krong Pa than in Huong Hoa (86.2% vs 39.3%). In the two districts, the prevalence of confirmed resistance was found in 69.0% and 1.2% of patients, respectively. In Thuan Bac district, we found intermediate prevalence of confirmed resistance. Treatment failure was not evidenced in any district. PPQ resistance was not evidenced. Confirmed resistance was associated to the persistence of parasites on day 28 and to 3.4-fold higher parasite density at diagnosis. The effectiveness of malaria control strategies was very high in the studied districts.ConclusionNo treatment failure has been observed in presence of high prevalence of ART resistance and in absence of PPQ resistance. K13(C580Y) was strongly associated to higher parasitemia at admission, on days 3 and 28. Slower parasite clearance was also observed in younger patients.

OC 8459 ASSESSMENT OF PARASITE CLEARANCE AFTER REPEATED TREATMENT WITH ARTESUNATE AMODIAQUINE, DIHYDROARTEMISININ-PIPERAQUINE, PYRONARIDINE-ARTESUNATE IN MALARIA PATIENTS IN BURKINA FASO

BackgroundReports from Southeast Asia showed delayed parasite clearance after treatment with known artemisinin-based combination therapies (ACTs), the first-line treatment for malaria. We then carried out a study in the framework of the WANECAM clinical trial to assess comparatively the parasite clearance time and rate from P. falciparum malaria patients repeatedly treated with the artesunate-amodiaquine (ASAQ), dihydroartemisinin-piperaquine (DHA-PQ) and artesunate-pyronaridine (PYR).MethodsA randomised, phase III/IV comparative, multicentre, open-label, parallel 3-arms trial was conducted in Banfora Health District area comparing the efficacy of a three-day regimen of DHA-PQ, PYR with ASAQ for the treatment of children (above 6 months) and adults with uncomplicated falciparum malaria. From August 2012 to December 2013, each randomised patient was followed up for 42 days over a period of two years. Treatment was directly observed, and blood smear samples were collected twice daily (12 hour±2 hour) until parasite clearance.The endpoints of the present sub-study were parasite clearance rate and time. The secondary endpoints included PCR-corrected and uncorrected cure rates.ResultsOut of 2843 screened patients, 763 were recruited for parasite clearance endpoint analyses. The median parasite clearance time (PCT) was 24.1 hour (2-sided 95% CI, 24.0 to 24.2 hour), 23.9 hour (2-sided 95% CI, 23.8 to 24.0 hour) and 24.2 hour (2-sided 95% CI, 24.1 to 24.5 hour), in PYR and DHA-PQ, respectively. The PCR-corrected efficacy rates were estimated at 99.8%; 99.7%; 99.9%, at day 28% and 99.3%; 99.7%–99.9% in PYR, ASAQ and DHA-PQ, respectively.ConclusionThe parasite clearance times were comparable among the three ACT arms of treatment and their efficacy was comparable and higher than 99%. There was no delay in parasite clearance time (PCT ≥72 hour).

Viability Screen of LOPAC1280 Reveals Tyrosine Kinase Inhibitor Tyrphostin A9 as a Novel Partner Drug for Artesunate Combinations To Target the Plasmodium falciparum Ring Stage.

The emergence of artemisinin-resistant Plasmodium falciparum poses a major threat to current frontline artemisinin combination therapies. Artemisinin resistance is widely associated with mutations in the P. falciparum Kelch13 (PfKelch13) propeller region, leading to delayed parasite clearance and increased survival of early-ring-stage parasites. There is therefore a need to discover novel drugs that are effective against artemisinin-resistant P. falciparum In view of this, our study aimed to identify compounds from the Library of Pharmacologically Active Compounds1280 (LOPAC1280) that could increase the efficacy of artesunate and be used as a potential partner drug for treatment against artemisinin-resistant falciparum malaria. By using a modified ring-stage survival assay, we performed a high-throughput screening of the activities of the 1,280 compounds from the LOPAC library in combination with artesunate against the P. falciparum IPC 5202 field isolate harboring the R539T mutation in the PfKelch13 propeller region. The potencies of the hits against both the IPC 5202 and CamWT_C580Y field isolates were determined through dose-dependent isobologram analyses; CamWT_C580Y has the more prevalent C580Y mutation characteristic of strains with artemisinin resistance. We identified tyrphostin A9 to have synergistic and additive activity against both parasite strains when dosed in combination with artesunate. These findings provide promising novel artesunate combinations that can target the P. falciparum artemisinin-resistant ring stage and insights that may aid in obtaining a better understanding of the mechanism involved in artemisinin resistance.

Plasmodium falciparum K13 expression associated with parasite clearance during artemisinin-based combination therapy.

Delayed parasite clearance and, consequently, reduced efficacy of artemisinin-based combination therapies have been linked with Plasmodium falciparum K13 gene SNPs in Southeast Asia. In Africa, significantly prolonged clearance has not yet been observed and the presently restricted variation in parasite clearance cannot be explained by K13 polymorphisms. Our aim was to study the in vivo pfK13 transcriptional response in patients treated with artemether-lumefantrine and explore whether the pfk13 transcripts can explain the patients' parasite clearance outcomes. A total of 47 Tanzanian children with microscopically confirmed uncomplicated P. falciparum malaria were hospitalized and received artemether-lumefantrine treatment (clinical trial ID: NCT00336375). RNA was extracted from venous blood samples collected before treatment initiation and at five more timepoints after treatment. cDNA was synthesized and pfk13 transcripts measured by real-time PCR. A wide range of pfk13 transcript variation was observed throughout all timepoints after artemether-lumefantrine treatment. Taking parasite clearance data together with the pfk13 transcripts profile, we observed a negative correlation inferring that pfk13 down-regulation is associated with longer parasite clearance time. The findings suggest that a reduced PfK13 transcriptional response may represent a first step towards artemisinin tolerance/resistance.

Association of mutations in the Plasmodium falciparum Kelch13 gene (Pf3D7_1343700) with parasite clearance rates after artemisinin-based treatments\u2014a WWARN individual patient data meta-analysis

BackgroundPlasmodium falciparum infections with slow parasite clearance following artemisinin-based therapies are widespread in the Greater Mekong Subregion. A molecular marker of the slow clearance phenotype has been identified: single genetic changes within the propeller region of the Kelch13 protein (pfk13; Pf3D7_1343700). Global searches have identified almost 200 different non-synonymous mutant pfk13 genotypes. Most mutations occur at low prevalence and have uncertain functional significance. To characterize the impact of different pfk13 mutations on parasite clearance, we conducted an individual patient data meta-analysis of the associations between parasite clearance half-life (PC1/2) and pfk13 genotype based on a large set of individual patient records from Asia and Africa.MethodsA systematic literature review following the PRISMA protocol was conducted to identify studies published between 2000 and 2017 which included frequent parasite counts and pfk13 genotyping. Four databases (Ovid Medline, PubMed, Ovid Embase, and Web of Science Core Collection) were searched. Eighteen studies (15 from Asia, 2 from Africa, and one multicenter study with sites on both continents) met inclusion criteria and were shared. Associations between the log transformed PC1/2 values and pfk13 genotype were assessed using multivariable regression models with random effects for study site.ResultsBoth the pfk13 genotypes and the PC1/2 were available from 3250 (95%) patients (n = 3012 from Asia (93%), n = 238 from Africa (7%)). Among Asian isolates, all pfk13 propeller region mutant alleles observed in five or more specific isolates were associated with a 1.5- to 2.7-fold longer geometric mean PC1/2 compared to the PC1/2 of wild type isolates (all p ≤ 0.002). In addition, mutant allele E252Q located in the P. falciparum region of pfk13 was associated with 1.5-fold (95%CI 1.4–1.6) longer PC1/2. None of the isolates from four countries in Africa showed a significant difference between the PC1/2 of parasites with or without pfk13 propeller region mutations.Previously, the association of six pfk13 propeller mutant alleles with delayed parasite clearance had been confirmed. This analysis demonstrates that 15 additional pfk13 alleles are associated strongly with the slow-clearing phenotype in Southeast Asia.ConclusionPooled analysis associated 20 pfk13 propeller region mutant alleles with the slow clearance phenotype, including 15 mutations not confirmed previously.

Current scenario and future strategies to fight artemisinin resistance.

Despite several setbacks in the fight against malaria such as insecticide and drug resistance as well as low efficacy of available vaccines, considerable success in reducing malaria burden has been achieved in the past decade. Artemisinins (ARTs and their combination therapies, ACTs), the current frontline drugs against uncomplicated malaria, rapidly kill plasmodial parasites and are non-toxic at short exposures. Though the exact mode of action remains unclear, the endoperoxide bridge, indispensable for ART activity, is thought to react with heme released from hemoglobin hydrolysis and generate free radicals that alkylate multiple protein targets, thereby disrupting proteostasis pathways. However, rapid development of ART resistance in recent years with no potential alternatives on the horizon threaten the elimination efforts. The Greater Mekong Subregion in South-East Asia continues to churn out mutants resistant to multiple ACTs and detected in increasingly expanding geographies. Extensive research on ART-resistant strains have identified a potential candidate Kelch13, crucial for mediating ART resistance. Parasites with mutations in the propeller domains of Plasmodium falciparum Kelch13 protein were shown to have enhanced phosphatidylinositol 3-kinase levels that were concomitant with delayed parasite clearance. Current research focused on understanding the mechanism of Kelch13-mediated ART resistance could provide better insights into Plasmodium resistome. This review covers the current proposed mechanisms of ART activity, resistance strategies adopted by the parasite in response to ACTs and possible future approaches to mitigate the spread of resistance from South-East Asia.

TACI Contributes to Plasmodium yoelii Host Resistance by Controlling T Follicular Helper Cell Response and Germinal Center Formation.

The delay in parasite-specific B cell development leaves people in malaria endemic areas vulnerable to repeated Plasmodium infections. Here, we investigated the role of transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI), a molecule involved in the generation of antigen-specific antibody secreting cells, in host response to non-lethal Plasmodium yoelii infection. We found that TACI deficiency not only resulted in higher peak parasitemia levels in P. yoelii challenged mice, but also led to a delay in parasite clearance and anti-P. yoelii Merozoite Surface Protein 1(C-terminal 19-kDa fragment [rMSP-119]) protein and anti-rMSP-119 and anti-P. yoelii IgG antibody development. There was also a delay in the generation of splenic high affinity antibody secreting cells that recognize rMSP-119 protein as compared to wild-type mice. Interestingly, coinciding with the delay in parasite clearance there was a delay in the resolution of T follicular helper (TFH) cell and germinal center (GC) B cell responses in TACI -/- mice. The persistence of TFH and GC B cells is likely a result of enhanced interaction between TFH and GC B cells because inducible costimulator ligand (ICOSL) expression was significantly higher on TACI -/- GC B cells than wild-type cells. The difference in the kinetics of GC reaction appeared to also impact the emergence of plasma cells (PC) because there was a delay in the generation of TACI -/- mice PC. Nevertheless, following the recovery from P. yoelii infection, TACI -/- and wild-type mice were both protected from a rechallenge infection. Establishment of protective B cell response was responsible for the resolution of parasitemia because B cells purified from recovered TACI -/- or wild-type mice were equally protective when introduced to naïve wild-type mice prior to P. yoelii challenge. Thus, despite the increased susceptibility of TACI -/- mice to P. yoelii infection and a delay in the development of protective antibody levels, TACI -/- mice are able to clear the infection and resist rechallenge infection.

High-level artemisinin-resistance with quinine co-resistance emerges in P. falciparum malaria under in vivo artesunate pressure

BackgroundHumanity has become largely dependent on artemisinin derivatives for both the treatment and control of malaria, with few alternatives available. A Plasmodium falciparum phenotype with delayed parasite clearance during artemisinin-based combination therapy has established in Southeast Asia, and is emerging elsewhere. Therefore, we must know how fast, and by how much, artemisinin-resistance can strengthen.MethodsP. falciparum was subjected to discontinuous in vivo artemisinin drug pressure by capitalizing on a novel model that allows for long-lasting, high-parasite loads. Intravenous artesunate was administered, using either single flash-doses or a 2-day regimen, to P. falciparum-infected humanized NOD/SCID IL-2Rγ−/−immunocompromised mice, with progressive dose increments as parasites recovered. The parasite’s response to artemisinins and other available anti-malarial compounds was characterized in vivo and in vitro.ResultsArtemisinin resistance evolved very rapidly up to extreme, near-lethal doses of artesunate (240 mg/kg), an increase of > 3000-fold in the effective in vivo dose, far above resistance levels reported from the field. Artemisinin resistance selection was reproducible, occurring in 80% and 41% of mice treated with flash-dose and 2-day regimens, respectively, and the resistance phenotype was stable. Measuring in vitro sensitivity proved inappropriate as an early marker of resistance, as IC50 remained stable despite in vivo resistance up to 30 mg/kg (ART-S: 10.7 nM (95% CI 10.2–11.2) vs. ART-R30: 11.5 nM (6.6–16.9), F = 0.525, p = 0.47). However, when in vivo resistance strengthened further, IC50 increased 10-fold (ART-R240 100.3 nM (92.9–118.4), F = 304.8, p < 0.0001), reaching a level much higher than ever seen in clinical samples. Artemisinin resistance in this African P. falciparum strain was not associated with mutations in kelch-13, casting doubt over the universality of this genetic marker for resistance screening. Remarkably, despite exclusive exposure to artesunate, full resistance to quinine, the only other drug sufficiently fast-acting to deal with severe malaria, evolved independently in two parasite lines exposed to different artesunate regimens in vivo, and was confirmed in vitro.ConclusionP. falciparum has the potential to evolve extreme artemisinin resistance and more complex patterns of multidrug resistance than anticipated. If resistance in the field continues to advance along this trajectory, we will be left with a limited choice of suboptimal treatments for acute malaria, and no satisfactory option for severe malaria.

Molecular assays for antimalarial drug resistance surveillance: A target product profile.

Antimalarial drug resistance is a major constraint for malaria control and elimination efforts. Artemisinin-based combination therapy is now the mainstay for malaria treatment. However, delayed parasite clearance following treatment with artemisinin derivatives has now spread in the Greater Mekong Sub region and may emerge or spread to other malaria endemic regions. This spread is of great concern for malaria control programmes, as no alternatives to artemisinin-based combination therapies are expected to be available in the near future. There is a need to strengthen surveillance systems for early detection and response to the antimalarial drug resistance threat. Current surveillance is mainly done through therapeutic efficacy studies; however these studies are complex and both time- and resource-intensive. For multiple common antimalarials, parasite drug resistance has been correlated with specific genetic mutations, and the molecular markers associated with antimalarial drug resistance offer a simple and powerful tool to monitor the emergence and spread of resistant parasites. Different techniques to analyse molecular markers associated with antimalarial drug resistance are available, each with advantages and disadvantages. However, procedures are not adequately harmonized to facilitate comparisons between sites. Here we describe the target product profiles for tests to analyse molecular markers associated with antimalarial drug resistance, discuss how use of current techniques can be standardised, and identify the requirements for an ideal product that would allow malaria endemic countries to provide useful spatial and temporal information on the spread of resistance.

Imidazolopiperazines Kill both Rings and Dormant Rings in Wild-Type and K13 Artemisinin-Resistant Plasmodium falciparum In Vitro.

ABSTRACTArtemisinin (ART) resistance has spread through Southeast Asia, posing a serious threat to the control and elimination of malaria. ART resistance has been associated with mutations in the Plasmodium falciparum kelch-13 (Pfk13) propeller domain. Phenotypically, ART resistance is defined as delayed parasite clearance in patients due to the reduced susceptibility of early ring-stage parasites to the active metabolite of ART dihydroartemisinin (DHA). Early rings can enter a state of quiescence upon DHA exposure and resume growth in its absence. These quiescent rings are referred to as dormant rings or DHA-pretreated rings (here called dormant rings). The imidazolopiperazines (IPZ) are a novel class of antimalarial drugs that have demonstrated efficacy in early clinical trials. Here, we characterized the stage of action of the IPZ GNF179 and evaluated its activity against rings and dormant rings in wild-type and ART-resistant parasites. Unlike DHA, GNF179 does not induce dormancy. We show that GNF179 is more rapidly cidal against schizonts than against ring and trophozoite stages. However, with 12 h of exposure, the compound effectively kills rings and dormant rings of both susceptible and ART-resistant parasites within 72 h. We further demonstrate that in combination with ART, GNF179 effectively prevents recrudescence of dormant rings, including those bearing pfk13 propeller mutations.

Assessing naturally acquired immune response and malaria treatment outcomes in Lagos, Nigeria

Background: There are emerging reports of poor efficacy of artemisinin-based combination treatment (ACT). However, mutations on the Kelch-13 gene marking delayed parasite clearance have no clinically defined relationship with ACT resistance across Africa. With increasing malaria control efforts, declining acquired immunity could be responsible for varying drug response profiles that may be dependent on levels of exposure to infections. To examine antibody responses against malaria and the influence on the efficacy of artemether-lumefantrine (AL), plasma samples were collected, prior to treatment, from individuals presenting with uncomplicated malaria. Methods: Participants were stratified into two groups: early (within 24 hours, N = 20) and late (between 48 – 72 hours, N = 30) parasite clearance after treatment, as determined by var gene acidic terminal sequence (varATS) polymerase chain reaction. Magnetic bead-based luminex assay was used to profile antibody responses specific to a panel of 21 Plasmodium falciparum sporozoite, merozoite and An. gambiae salivary antigens. Results: Median fluorescence intensity (MFI) of the antibodies was highest against glutamate-rich protein (GLURP-R0) and lowest against merozoite surface protein (MSP2) antigen. Analysis showed a positive correlation between expression of immunity and age of individuals (P = 0.023). However, there was no association between parasite density and antibody responses, except a significant positive relationship with reticulocyte binding protein-like homologue 5 (Rh5), P = 0.047; Plasmodium exported protein (Hyp2), P = 0.037 and merozoite surface protein 11 (H103), P = 0.038. Though higher levels of antibodies against erythrocyte binding antigens (EBA 140 and 175), MSP1.19, GLURP, circumsporozoite protein (CSP) and Rh4.2 were observed in individuals who recorded early parasite clearance, there was no significant difference in antibody responses in the early and late parasitological response groups. Conclusions: Characterization of additional markers in larger populations is required to reveal potential immunological correlates of drug efficacy.

Artemisinin-Resistant Plasmodium falciparum with High Survival Rates, Uganda, 2014-2016.

Because ≈90% of malaria cases occur in Africa, emergence of artemisinin-resistant Plasmodium falciparum in Africa poses a serious public health threat. To assess emergence of artemisinin-resistant parasites in Uganda during 2014–2016, we used the recently developed ex vivo ring-stage survival assay, which estimates ring-stage–specific P. falciparum susceptibility to artemisinin. We conducted 4 cross-sectional surveys to assess artemisinin sensitivity in Gulu, Uganda. Among 194 isolates, survival rates (ratio of viable drug-exposed parasites to drug-nonexposed controls) were high (>10%) for 4 isolates. Similar rates have been closely associated with delayed parasite clearance after drug treatment and are considered to be a proxy for the artemisinin-resistant phenotype. Of these, the PfKelch13 mutation was observed in only 1 isolate, A675V. Population genetics analysis suggested that these possibly artemisinin-resistant isolates originated in Africa. Large-scale surveillance of possibly artemisinin-resistant parasites in Africa would provide useful information about treatment outcomes and help regional malaria control.

Prevalence of Plasmodium falciparum delayed clearance associated polymorphisms in adaptor protein complex 2 mu subunit (pfap2mu) and ubiquitin specific protease 1 (pfubp1) genes in Ghanaian isolates

BackgroundPlasmodium falciparum delayed clearance with the use of artemisinin-based combination therapy (ACTs) has been reported in some African countries. Single nucleotide polymorphisms (SNPs) in two genes, P. falciparum adaptor protein complex 2 mu subunit (pfap2mu) and ubiquitin specific protease 1 (pfubp1), have been linked to delayed clearance with ACT use in Kenya and recurrent imported malaria in Britain. With over 12 years of ACT use in Ghana, this study investigated the prevalence of SNPs in the pfap2mu and pfubp1 in Ghanaian clinical P. falciparum isolates to provide baseline data for antimalarial drug resistance surveillance in the country.MethodsFilter paper blood blots collected in 2015–2016 from children aged below 9 years presenting with uncomplicated malaria at hospitals in three sentinel sites Begoro, Cape Coast and Navrongo were used. Parasite DNA was extracted from 120 samples followed by nested polymerase chain reaction (nPCR). Sanger sequencing was performed to detect and identify SNPs in pfap2mu and pfubp1 genes.ResultsIn all, 11.1% (9/81) of the isolates carried the wildtype genotypes for both genes. A total of 164 pfap2mu mutations were detected in 67 isolates whilst 271 pfubp1 mutations were observed in 72 isolates. The majority of the mutations were non-synonymous (NS): 78% (128/164) for pfap2mu and 92.3% (250/271) for pfubp1. Five unique samples had a total of 215 pfap2mu SNPs, ranging between 15 and 63 SNPs per sample. Genotypes reportedly associated with ART resistance detected in this study included pfap2mu S160N (7.4%, 6/81) and pfubp1 E1528D (7.4%, 6/81) as well as D1525E (4.9%, 4/81). There was no significant difference in the prevalence of the SNPs between the three ecologically distinct study sites (pfap2mu: χ2 = 6.905, df = 2, P = 0.546; pfubp1: χ2 = 4.883, df = 2, P = 0.769).ConclusionsThe detection of pfap2mu and pfubp1 genotypes associated with ACT delayed parasite clearance is evidence of gradual nascent emergence of resistance in Ghana. The results will serve as baseline data for surveillance and the selection of the genotypes with drug pressure over time. The pfap2mu S160N, pfubp1 E1528D and D1525E must be monitored in Ghanaian isolates in ACT susceptibility studies, especially when cure rates of ACTs, particularly AL, is less than 100%.

Efficacy of artesunate\u2013amodiaquine, dihydroartemisinin\u2013piperaquine and artemether\u2013lumefantrine for the treatment of uncomplicated Plasmodium falciparum malaria in Maradi, Niger

BackgroundMalaria endemic countries need to assess efficacy of anti-malarial treatments on a regular basis. Moreover, resistance to artemisinin that is established across mainland South-East Asia represents today a major threat to global health. Monitoring the efficacy of artemisinin-based combination therapies is of paramount importance to detect as early as possible the emergence of resistance in African countries that toll the highest burden of malaria morbidity and mortality.MethodsA WHO standard protocol was used to assess efficacy of the combinations artesunate–amodiaquine (AS–AQ Winthrop®), dihydroartemisinin–piperaquine (DHA–PPQ, Eurartesim®) and artemether–lumefantrine (AM–LM, Coartem®) taken under supervision and respecting pharmaceutical recommendations. The study enrolled for each treatment arm 212 children aged 6–59 months living in Maradi (Niger) and suffering with uncomplicated falciparum malaria. The Kaplan–Meier 42-day PCR-adjusted cure rate was the primary outcome. A standardized parasite clearance estimator was used to assess delayed parasite clearance as surrogate maker of suspected artemisinin resistance.ResultsNo early treatment failures were found in any of the study treatment arms. The day-42 PCR-adjusted cure rate estimates were 99.5, 98.4 and 99.0% in the AS–AQ, DHA–PPQ and AM–LM arms, respectively. The reinfection rate (expressed also as Kaplan–Meier estimates) was higher in the AM–LM arm (32.4%) than in the AS–AQ (13.8%) and the DHA–PPQ arm (24.9%). The parasite clearance rate constant was 0.27, 0.26 and 0.25 per hour for AS–AQ, DHA–PPQ and AM–LM, respectively.ConclusionsAll the three treatments evaluated largely meet WHO criteria (at least 95% efficacy). AS–AQ and AL–LM may continue to be used and DHA–PPQ may be also recommended as first-line treatment for uncomplicated falciparum malaria in Maradi. The parasite clearance rate were consistent with reference values indicating no suspected artemisinin resistance. Nevertheless, the monitoring of anti-malarial drug efficacy should continue.Trial registration details Registry number at ClinicalTrial.gov: NCT01755559

A Novel Single-Nucleotide Polymorphism Loop Mediated Isothermal Amplification Assay for Detection of Artemisinin-Resistant Plasmodium falciparum Malaria.

BackgroundArtemisinin-resistant malaria (ARM) remains a significant threat to malaria elimination. In the Greater Mekong subregion, the prevalence of ARM in certain regions has reached greater than 90%. Artemisinin-resistant malaria is clinically identified by delayed parasite clearance and has been associated with mutations in the propeller domain of the kelch 13 gene. C580Y is the most prevalent mutation. The detection of ARM currently relies on labor-intensive and time-consuming methods such as clinical phenotyping or in vitro susceptibility testing.MethodsWe developed a novel single-nucleotide polymorphism loop mediated isothermal amplification (SNP-LAMP) test method for the detection of the C580Y mutation using a novel primer design strategy.ResultsThe SNP-LAMP was 90.0% sensitive (95% confidence interval [CI], 66.9–98.3) and 91.9% specific (95% CI, 82.6–96.7) without knowledge of the parasite load and was 100% sensitive (95% CI, 79.9–100) and 97.3% specific (95% CI, 89.7–99.5) when the parasitemia was within the assay dynamic range. Tests with potential application near-to-patient such as SNP-LAMP may be deployed in low- and middle-income and developed countries.ConclusionsSingle-nucleotide polymorphism LAMP can serve as a surveillance tool and guide treatment algorithms for ARM in a clinically relevant time frame, prevent unnecessary use of additional drugs that may drive additional resistance, and avoid longer treatment regimens that cause toxicity for the patient.

Artemisinin resistance without pfkelch13 mutations in Plasmodium falciparum isolates from Cambodia

BackgroundArtemisinin resistance is associated with delayed parasite clearance half-life in vivo and correlates with ring-stage survival under dihydroartemisinin in vitro. Both phenotypes are associated with mutations in the PF3D7_1343700 pfkelch13 gene. Recent spread of artemisinin resistance and emerging piperaquine resistance in Southeast Asia show that artemisinin combination therapy, such as dihydroartemisinin–piperaquine, are losing clinical effectiveness, prompting investigation of drug resistance mechanisms and development of strategies to surmount emerging anti-malarial resistance.MethodsSixty-eight parasites isolates with in vivo clearance data were obtained from two Tracking Resistance to Artemisinin Collaboration study sites in Cambodia, culture-adapted, and genotyped for pfkelch13 and other mutations including pfmdr1 copy number; and the RSA0–3h survival rates and response to antimalarial drugs in vitro were measured for 36 of these isolates.ResultsAmong these 36 parasites one isolate demonstrated increased ring-stage survival for a PfKelch13 mutation (D584V, RSA0–3h = 8%), previously associated with slow clearance but not yet tested in vitro. Several parasites exhibited increased ring-stage survival, yet lack pfkelch13 mutations, and one isolate showed evidence for piperaquine resistance.ConclusionsThis study of 68 culture-adapted Plasmodium falciparum clinical isolates from Cambodia with known clearance values, associated the D584V PfKelch13 mutation with increased ring-stage survival and identified parasites that lack pfkelch13 mutations yet exhibit increased ring-stage survival. These data suggest mutations other than those found in pfkelch13 may be involved in conferring artemisinin resistance in P. falciparum. Piperaquine resistance was also detected among the same Cambodian samples, consistent with reports of emerging piperaquine resistance in the field. These culture-adapted parasites permit further investigation of mechanisms of both artemisinin and piperaquine resistance and development of strategies to prevent or overcome anti-malarial resistance.

Absence of association between polymorphisms in the K13 gene and the presence of Plasmodium falciparum parasites at day 3 after treatment with artemisinin derivatives in Senegal.

In 2006, the Senegalese National Malaria Control Programme recommended artemisinin-based combination therapy as first-line treatment for uncomplicated malaria. In addition, intravenous (i.v.) injection of artesunate and artemether has gradually replaced quinine for the treatment of severe malaria. Mutations in the propeller domain of the Kelch 13 gene (K13-propeller, PF3D71343700), such as Y493H, R539T, I543T and C580Y, were recently associated with in vivo and in vitro resistance to artemisinin in Southeast Asia. However, these mutations were not identified in Africa. In total, 181 isolates of Plasmodium falciparum from 161 patients from Dakar, Senegal, were collected between August 2015 and January 2016. The K13-propeller gene of the isolates was sequenced. A search for non-synonymous mutations in the propeller region of K13 was performed in the 181 isolates collected from Dakar from 2015 to 2016. Three synonymous mutations were detected (D464D, C469C and R471R). Of 119 patients treated with i.v. artesunate or intramuscular artemether followed by artemether/lumefantrine, 9 patients were still parasitaemic on Day 3. Parasites from these nine patients were wild-type for K13-propeller. None of the polymorphisms known to be involved in artemisinin resistance in Asia were detected. These results suggest that K13 is not the best predictive marker for artemisinin resistance in Africa. More isolates from clinical failure cases or patients with delayed parasite clearance after treatment with artemisinin derivatives are necessary to identify new molecular markers.

Plasmodium falciparum K13 Mutations Differentially Impact Ozonide Susceptibility and Parasite Fitness In Vitro.

ABSTRACTThe emergence and spread in Southeast Asia of Plasmodium falciparum resistance to artemisinin (ART) derivatives, the cornerstone of first-line artemisinin-based combination therapies (ACTs), underscore the urgent need to identify suitable replacement drugs. Discovery and development efforts have identified a series of ozonides with attractive chemical and pharmacological properties that are being touted as suitable replacements. Partial resistance to ART, defined as delayed parasite clearance in malaria patients treated with an ART derivative or an ACT, has been associated with mutations in the P. falciparum K13 gene. In light of reports showing that ART derivatives and ozonides share similar modes of action, we have investigated whether parasites expressing mutant K13 are cross-resistant to the ozonides OZ439 (artefenomel) and OZ227 (arterolane). This work used a panel of culture-adapted clinical isolates from Cambodia that were genetically edited to express variant forms of K13. Phenotypic analyses employed ring-stage survival assays (ring-stage survival assay from 0 to 3 h [RSA0–3h]), whose results have earlier been shown to correlate with parasite clearance rates in patients. Our results document cross-resistance between OZ277 and dihydroartemisinin (DHA), a semisynthetic derivative of ART, in parasites carrying the K13 mutations C580Y, R539T, and I543T. For OZ439, we observed cross-resistance only for parasites that carried the rare K13 I543T mutation, with no evidence of cross-resistance afforded by the prevalent C580Y mutation. Mixed-culture competition experiments with isogenic lines carrying modified K13 revealed variable growth deficits depending on the K13 mutation and parasite strain and provide a rationale for the broad dissemination of the fitness-neutral K13 C580Y mutation throughout strains currently circulating in Southeast Asia.

Therapeutic efficacy and artemisinin resistance in northern Myanmar: evidence from in vivo and molecular marker studies

BackgroundIn Myanmar, three types of artemisinin-based combination therapy (ACT) are recommended as first-line treatment of uncomplicated falciparum malaria: artemether–lumefantrine (AL), artesunate–mefloquine (AS + MQ), and dihydroartemisinin–piperaquine (DP). Resistance to both artemisinins and ACT partner drugs has been reported from the Greater Mekong Sub-region, and regular efficacy monitoring of the recommended ACT is conducted in Myanmar. This paper reports on results from studies to monitor the efficacy of the three forms of ACT in sentinel sites in northern Myanmar, and investigations of mutations in the Kelch13 (k13) propeller domain.MethodsSeven therapeutic efficacy studies were conducted in 2011–12 and 2014 in three sentinel sites in Myanmar (Tamu, Muse, Tabeikkyin). Three studies were done for the evaluation of AL (204 patients), two studies for AS + MQ (119 patients) and two studies for DP (147 patients). These studies were done according to 2009 standard WHO protocol. Polymorphisms in the k13 propeller domain were examined in dried blood spots collected on day 0. The primary endpoint was adequate clinical and parasitological response (ACPR) on day 28 for AL and on day 42 for DP and AS + MQ, corrected to exclude re-infection using polymerase chain reaction (PCR) genotyping. Safety data were collected through self-reporting.ResultsPCR-corrected ACPR was 97.2–100% for AL, 98.6–100% for AS + MQ and 100% for DP across the study sites and years. All studies found a prevalence of k13 mutations (>440) above 23% in the day-0 samples. The F446I mutation was the most common mutation, making up 66.0% of the mutations found. Seven out of nine day-3 positive patients were infected with k13 wild type parasites. The remaining two cases with day-3 parasitaemia had the P574L mutation.ConclusionsThe efficacy of AL, AS + MQ and DP remains high in northern Myanmar despite widespread evidence of k13 mutations associated with delayed parasite clearance. This study showed that already in 2012 there was a high frequency of k13 mutations in Myanmar on the border with India. The high efficacy of the recommended ACT gives confidence in the continued recommendation of the use of these treatments in Myanmar.Trial registration numbers ACTRN12611001245987 (registered 06-12-2011) and ACTRN12614000216617 (registered 28-02-2014)