Prefoldins are novel regulators of the unfolded protein response in artemisinin resistant P. falciparum malaria.

Emerging Artemisinin (ART) resistance in Plasmodium falciparum (Pf) poses challenges for discovery of novel drugs to tackle ART resistant parasites. Concentrated efforts towards ART resistance mechanism indicated a strong molecular link of ART resistance with up-regulated expression of unfolded protein response pathways involving Prefoldins (PFDs). However, a complete characterization of PFDs as molecular players taking part in ART resistance mechanism, and discovery of small molecule inhibitors to block this process have not been identified to date. Here, we functionally characterized all Pf Prefoldin subunits (PFD1-6), and established a causative role played by PFDs in ART resistance by demonstrating their expression in intra-erythrocytic parasites along with their interactions with Kelch13 protein through immunoprecipitation coupled MS/MS analysis. Systematic biophysical interaction analysis between all subunits of PFDs revealed their potential to form a complex. The role of PFDs in ART resistance was confirmed in orthologous yeast PFD6 mutants, where PfPFD6 expression in yeast mutants reverted phenotype to ART resistance. We identified an FDA approved drug 'Biperiden' that restricts the formation of Prefoldin complex and inhibits its interaction with its key parasite protein substrates, MSP-1 and α-tubulin-I. Moreover, Biperiden treatment inhibits the parasite growth in ART sensitive Pf3D7 and resistant Pf3D7k13R539T strains. Ring survival assays that are clinically relevant to analyse ART resistance in Pf3D7k13R539T parasites demonstrate the potency of BPD to inhibit growth of survivor parasites. Overall, our study provides first evidence towards the role of PfPFDs in ART resistance mechanism, and opens new avenues for the management of resistant parasite.

Evaluation of Dihydroartemisinin-Piperaquine Efficacy and Molecular Markers in Uncomplicated Falciparum Patients: A Study across Binh Phuoc and Dak Nong, Vietnam.

Background and Objectives: Malaria continues to be a significant global health challenge. The efficacy of artemisinin-based combination therapies (ACTs) has declined in many parts of the Greater Mekong Subregion, including Vietnam, due to the spread of resistant malaria strains. This study was conducted to assess the efficacy of the Dihydroartemisinin (DHA)-Piperaquine (PPQ) regimen in treating uncomplicated falciparum malaria and to conduct molecular surveillance of antimalarial drug resistance in Binh Phuoc and Dak Nong provinces. Materials and Methods: The study included 63 uncomplicated malaria falciparum patients from therapeutic efficacy studies (TES) treated following the WHO treatment guidelines (2009). Molecular marker analysis was performed on all 63 patients. Methods encompassed Sanger sequencing for pfK13 mutations and quantitative real-time PCR for the pfpm2 gene. Results: This study found a marked decrease in the efficacy of the DHA-PPQ regimen, with an increased rate of treatment failures at two study sites. Genetic analysis revealed a significant presence of pfK13 mutations and pfpm2 amplifications, indicating emerging resistance to artemisinin and its partner drug. Conclusions: The effectiveness of the standard DHA-PPQ regimen has sharply declined, with rising treatment failure rates. This decline necessitates a review and possible revision of national malaria treatment guidelines. Importantly, molecular monitoring and clinical efficacy assessments together provide a robust framework for understanding and addressing detection drug resistance in malaria.

PfHDAC1 is an essential regulator of P. falciparum asexual proliferation and host cell invasion genes with a dynamic genomic occupancy responsive to artemisinin stress.

Plasmodium falciparum, the deadly protozoan parasite responsible for malaria, has a tightly regulated gene expression profile closely linked to its intraerythrocytic development cycle. Epigenetic modifiers of the histone acetylation code have been identified as key regulators of the parasite's transcriptome but require further investigation. In this study, we map the genomic distribution of Plasmodium falciparum histone deacetylase 1 (PfHDAC1) across the erythrocytic asexual development cycle and find it has a dynamic occupancy over a wide array of developmentally relevant genes. Overexpression of PfHDAC1 results in a progressive increment in parasite load over consecutive rounds of the asexual infection cycle and is associated with enhanced gene expression of multiple families of host cell invasion factors (merozoite surface proteins, rhoptry proteins, etc.) and with increased merozoite invasion efficiency. With the use of class-specific inhibitors, we demonstrate that PfHDAC1 activity in parasites is crucial for timely intraerythrocytic development. Interestingly, overexpression of PfHDAC1 results in decreased sensitivity to frontline-drug dihydroartemisinin in parasites. Furthermore, we identify that artemisinin exposure can interfere with PfHDAC1 abundance and chromatin occupancy, resulting in enrichment over genes implicated in response/resistance to artemisinin. Finally, we identify that dihydroartemisinin exposure can interrupt the in vitro catalytic deacetylase activity and post-translational phosphorylation of PfHDAC1, aspects that are crucial for its genomic function. Collectively, our results demonstrate PfHDAC1 to be a regulator of critical functions in asexual parasite development and host invasion, which is responsive to artemisinin exposure stress and deterministic of resistance to it. IMPORTANCE: Malaria is a major public health problem, with the parasite Plasmodium falciparum causing most of the malaria-associated mortality. It is spread by the bite of infected mosquitoes and results in symptoms such as cyclic fever, chills, and headache. However, if left untreated, it can quickly progress to a more severe and life-threatening form. The World Health Organization currently recommends the use of artemisinin combination therapy, and it has worked as a gold standard for many years. Unfortunately, certain countries in southeast Asia and Africa, burdened with a high prevalence of malaria, have reported cases of drug-resistant infections. One of the major problems in controlling malaria is the emergence of artemisinin resistance. Population genomic studies have identified mutations in the Kelch13 gene as a molecular marker for artemisinin resistance. However, several reports thereafter indicated that Kelch13 is not the main mediator but rather hinted at transcriptional deregulation as a major determinant of drug resistance. Earlier, we identified PfGCN5 as a global regulator of stress-responsive genes, which are known to play a central role in artemisinin resistance generation. In this study, we have identified PfHDAC1, a histone deacetylase as a cell cycle regulator, playing an important role in artemisinin resistance generation. Taken together, our study identified key transcriptional regulators that play an important role in artemisinin resistance generation.

Day 3 parasitemia and Plasmodium falciparum Kelch 13 mutations among uncomplicated malaria patients treated with artemether-lumefantrine in Adjumani district, Uganda.

Artemisinin resistance threatens malaria control and elimination efforts globally. Recent studies have reported the emergence of Plasmodium falciparum parasites tolerant to artemisinin agents in sub-Saharan Africa, including Uganda. The current study assessed the day 3 parasite clearance and its correlation with P. falciparum K13 propeller gene (pfkelch13) mutations in P. falciparum parasites isolated from patients with uncomplicated malaria under artemether-lumefantrine (AL) treatment. This study enrolled 100 P. falciparum-positive patients to whom AL was prescribed between 09/September/2022 and 06/November/2022. Blood samples were collected in EDTA tubes before treatment initiation (day 0) and on day 3. Parasitemia was assessed by microscopy from blood smears and quantitative polymerase chain reaction (qPCR) from the DNA extracted. The day 0 parasite K13 gene was sequenced using Sanger sequencing. Sequence data were analysed using MEGA version 11 software. The data were analysed using STATA version 15, and the Mann‒Whitney U test was used to compare PCR parasite clearance on day 3 using the comparative CT value method and pfkelch13 mutations. The prevalence of day 3 parasitaemia was 24% (24/100) by microscopy and 63% (63/100) by qPCR from the AL-treated patients. P. falciparum K13-propeller gene polymorphism was detected in 18.8% (15/80) of the day 0 DNA samples. The K13 mutations found were C469Y, 12.5% (10/80); A675V, 2.5% (2/80); A569S, 1.25%, (1/80), A578S, 1.25%, (1/80) and; F491S, 1.25%, (1/80) a new allele not reported anywhere. The C469Y mutation, compared to the wild-type, was associated with delayed parasite clearance p=0.0278, Hodges-Lehmann estimation 3.2108 on the log scale, (95%CI 1.7076, 4.4730). There was a high prevalence of day 3 P. falciparum among malaria patients treated using artemether-lumefantrine. We conclude that the K13 mutation associated with artemisinin resistance by P. falciparum is present in Adjumani district, Uganda. This necessitates regular surveillance of the effectiveness and efficacy of artemether-lumefantrine in the country.

Detection of novel Plasmodium falciparum coronin gene mutations in a recrudescent ACT-treated patient in South-Western Nigeria.

Background: Routine surveillance for antimalarial drug resistance is critical to sustaining the efficacy of artemisinin-based Combination Therapies (ACTs). Plasmodium falciparum kelch-13 (Pfkelch-13) and non-Pfkelch-13 artemisinin (ART) resistance-associated mutations are uncommon in Africa. We investigated polymorphisms in Plasmodium falciparum actin-binding protein (Pfcoronin) associated with in vivo reduced sensitivity to ART in Nigeria. Methods: Fifty-two P. falciparum malaria subjects who met the inclusion criteria were followed up in a 28-day therapeutic efficacy study of artemether-lumefantrine in Lagos, Nigeria. Parasite detection was done by microscopy and molecular diagnostic approaches involving PCR amplification of genes for Pf18S rRNA, varATS, telomere-associated repetitive elements-2 (TARE-2). Pfcoronin and Pfkelch-13 genes were sequenced bi-directionally while clonality of infections was determined using 12 neutral P. falciparum microsatellite loci and msp2 analyses. Antimalarial drugs (sulfadoxine-pyrimethamine, amodiaquine, chloroquine and some quinolones) resistance variants (DHFR_51, DHFR_59, DHFR_108, DHFR_164, MDR1_86, MDR1_184, DHPS_581 and DHPS_613) were genotyped by high-resolution melting (HRM) analysis. Results: A total of 7 (26.92%) cases were identified either as early treatment failure, late parasitological failure or late clinical failure. Of the four post-treatment infections identified as recrudescence by msp2 genotypes, only one was classified as recrudescence by multilocus microsatellites genotyping. Microsatellite analysis revealed no significant difference in the mean allelic diversity, He, (P = 0.19, Mann-Whitney test). Allele sizes and frequency per locus implicated one isolate. Genetic analysis of this isolate identified two new Pfcoronin SNVs (I68G and L173F) in addition to the P76S earlier reported. Linkage-Disequilibrium as a standardized association index, IAS, between multiple P. falciparum loci revealed significant LD (IAS = 0.2865, P=0.02, Monte-Carlo simulation) around the neutral microsatellite loci. The pfdhfr/pfdhps/pfmdr1 drug resistance-associated haplotypes combinations, (108T/N/51I/164L/59R/581G/86Y/184F), were observed in two samples. Conclusion: Pfcoronin mutations identified in this study, with potential to impact parasite clearance, may guide investigations on emerging ART tolerance in Nigeria, and West African endemic countries.

Molecular markers of artemisinin resistance during falciparum malaria elimination in Eastern Myanmar.

BACKGROUND: Artemisinin resistance in Plasmodium falciparum threatens global malaria elimination efforts. To contain and then eliminate artemisinin resistance in Eastern Myanmar a network of community-based malaria posts was instituted and targeted mass drug administration (MDA) with dihydroartemisinin-piperaquine (three rounds at monthly intervals) was conducted. The prevalence of artemisinin resistance during the elimination campaign (2013-2019) was characterized. METHODS: Throughout the six-year campaign Plasmodium falciparum positive blood samples from symptomatic patients and from cross-sectional surveys were genotyped for mutations in kelch-13-a molecular marker of artemisinin resistance. RESULT: The program resulted in near elimination of falciparum malaria. Of 5162 P. falciparum positive blood samples genotyped, 3281 (63.6%) had K13 mutations. The prevalence of K13 mutations was 73.9% in 2013 and 64.4% in 2019. Overall, there was a small but significant decline in the proportion of K13 mutants (p < 0.001). In the MDA villages there was no significant change in the K13 proportions before and after MDA. The distribution of different K13 mutations changed substantially; F446I and P441L mutations increased in both MDA and non-MDA villages, while most other K13 mutations decreased. The proportion of C580Y mutations fell from 9.2% (43/467) before MDA to 2.3% (19/813) after MDA (p < 0.001). Similar changes occurred in the 487 villages where MDA was not conducted. CONCLUSION: The malaria elimination program in Kayin state, eastern Myanmar, led to a substantial reduction in falciparum malaria. Despite the intense use of artemisinin-based combination therapies, both in treatment and MDA, this did not select for artemisinin resistance.

Drug-induced stress mediates Plasmodium falciparum ring-stage growth arrest and reduces in vitro parasite susceptibility to artemisinin.

During blood-stage infection, Plasmodium falciparum parasites are constantly exposed to a range of extracellular stimuli, including host molecules and drugs such as artemisinin derivatives, the mainstay of artemisinin-based combination therapies currently used as first-line treatment worldwide. Partial resistance of P. falciparum to artemisinin has been associated with mutations in the propeller domain of the Pfkelch13 gene, resulting in a fraction of ring stages that are able to survive exposure to artemisinin through a temporary growth arrest. Here, we investigated whether the growth arrest in ring-stage parasites reflects a general response to stress. We mimicked a stressful environment in vitro by exposing parasites to chloroquine or dihydroartemisinin (DHA). We observed that early ring-stage parasites pre-exposed to a stressed culture supernatant exhibited a temporary growth arrest and a reduced susceptibility to DHA, as assessed by the ring-stage survival assay, irrespective of their Pfkelch13 genotype. These data suggest that temporary growth arrest of early ring stages may be a constitutive, Pfkelch13-independent survival mechanism in P. falciparum.IMPORTANCEPlasmodium falciparum ring stages have the ability to sense the extracellular environment, regulate their growth, and enter a temporary growth arrest state in response to adverse conditions such as drug exposure. This temporary growth arrest results in reduced susceptibility to artemisinin in vitro. The signal responsible for this process is thought to be small molecules (less than 3 kDa) released by stressed mature-stage parasites. These data suggest that Pfkelch13-dependent artemisinin resistance and the growth arrest phenotype are two complementary but unrelated mechanisms of ring-stage survival in P. falciparum. This finding provides new insights into the field of P. falciparum antimalarial drug resistance by highlighting the extracellular compartment and cellular communication as an understudied mechanism.

Temporal and spatial dynamics of Plasmodium falciparum clonal lineages in Guyana.

Plasmodium parasites, the causal agents of malaria, are eukaryotic organisms that obligately undergo sexual recombination within mosquitoes. However, in low transmission settings where most mosquitoes become infected with only a single parasite clone, parasites recombine with themselves, and the clonal lineage is propagated rather than broken up by outcrossing. We investigated whether stochastic/neutral factors drive the persistence and abundance of Plasmodium falciparum clonal lineages in Guyana, a country with relatively low malaria transmission, but the only setting in the Americas in which an important artemisinin resistance mutation (pfk13 C580Y) has been observed. To investigate whether this clonality was potentially associated with the persistence and spatial spread of the mutation, we performed whole genome sequencing on 1,727 Plasmodium falciparum samples collected from infected patients across a five-year period (2016-2021). We characterized the relatedness between each pair of monoclonal infections (n=1,409) through estimation of identity by descent (IBD) and also typed each sample for known or candidate drug resistance mutations. A total of 160 clones (mean IBD ≥ 0.90) were circulating in Guyana during the study period, comprising 13 highly related clusters (mean IBD ≥ 0.40). In the five-year study period, we observed a decrease in frequency of a mutation associated with artemisinin partner drug (piperaquine) resistance (pfcrt C350R) and limited co-occurence of pfcrt C350R with duplications of plasmepsin 2/3, an epistatic interaction associated with piperaquine resistance. We additionally report polymorphisms exhibiting evidence of selection for drug resistance or other phenotypes and reported a novel pfk13 mutation (G718S) as well as 61 nonsynonymous substitutions that increased markedly in frequency. However, P. falciparum clonal dynamics in Guyana appear to be largely driven by stochastic factors, in contrast to other geographic regions. The use of multiple artemisinin combination therapies in Guyana may have contributed to the disappearance of the pfk13 C580Y mutation.

Molecular surveillance of Kelch 13 polymorphisms in Plasmodium falciparum isolates from Kenya and Ethiopia.

BACKGROUND: Timely molecular surveillance of Plasmodium falciparum kelch 13 (k13) gene mutations is essential for monitoring the emergence and stemming the spread of artemisinin resistance. Widespread artemisinin resistance, as observed in Southeast Asia, would reverse significant gains that have been made against the malaria burden in Africa. The purpose of this study was to assess the prevalence of k13 polymorphisms in western Kenya and Ethiopia at sites representing varying transmission intensities between 2018 and 2022. METHODS: Dried blood spot samples collected through ongoing passive surveillance and malaria epidemiological studies, respectively, were investigated. The k13 gene was genotyped in P. falciparum isolates with high parasitaemia: 775 isolates from four sites in western Kenya (Homa Bay, Kakamega, Kisii, and Kombewa) and 319 isolates from five sites across Ethiopia (Arjo, Awash, Gambella, Dire Dawa, and Semera). DNA sequence variation and neutrality were analysed within each study site where mutant alleles were detected. RESULTS: Sixteen Kelch13 haplotypes were detected in this study. Prevalence of nonsynonymous k13 mutations was low in both western Kenya (25/783, 3.19%) and Ethiopia (5/319, 1.57%) across the study period. Two WHO-validated mutations were detected: A675V in three isolates from Kenya and R622I in four isolates from Ethiopia. Seventeen samples from Kenya carried synonymous mutations (2.17%). No synonymous mutations were detected in Ethiopia. Genetic variation analyses and tests of neutrality further suggest an excess of low frequency polymorphisms in each study site. Fu and Li's F test statistic in Semera was 0.48 (P > 0.05), suggesting potential population selection of R622I, which appeared at a relatively high frequency (3/22, 13.04%). CONCLUSIONS: This study presents an updated report on the low frequency of k13 mutations in western Kenya and Ethiopia. The WHO-validated R622I mutation, which has previously only been reported along the north-west border of Ethiopia, appeared in four isolates collected from eastern Ethiopia. The rapid expansion of R622I across Ethiopia signals the need for enhanced monitoring of the spread of drug-resistant P. falciparum parasites in East Africa. Although ACT remains currently efficacious in the study areas, continued surveillance is necessary to detect early indicators of artemisinin partial resistance.

Assessment of Plasmodium falciparum drug resistance associated molecular markers in Mandla, Madhya Pradesh, India.

BACKGROUND: Resistance against artemisinin-based combination therapy is one of the challenges to malaria control and elimination globally. Mutations in different genes (Pfdhfr, Pfdhps, Pfk-13 and Pfmdr1) confer resistance to artesunate and sulfadoxine-pyrimethamine (AS + SP) were analysed from Mandla district, Madhya Pradesh, to assess the effectiveness of the current treatment regimen against uncomplicated Plasmodium falciparum. METHODS: Dried blood spots were collected during the active fever survey and mass screening and treatment activities as part of the Malaria Elimination Demonstration Project (MEDP) from 2019 to 2020. Isolated DNA samples were used to amplify the Pfdhfr, Pfdhps, Pfk13 and Pfmdr1 genes using nested PCR and sequenced for mutation analysis using the Sanger sequencing method. RESULTS: A total of 393 samples were subjected to PCR amplification, sequencing and sequence analysis; 199, 215, 235, and 141 samples were successfully sequenced for Pfdhfr, Pfdhps, Pfk13, Pfmdr1, respectively. Analysis revealed that the 53.3% double mutation (C59R, S108N) in Pfdhfr, 89.3% single mutation (G437A) in Pfdhps, 13.5% single mutants (N86Y), and 51.1% synonymous mutations in Pfmdr1 in the study area. Five different non-synonymous and two synonymous point mutations found in Pfk13, which were not associated to artemisinin resistance. CONCLUSION: The study has found that mutations linked to SP resistance are increasing in frequency, which may reduce the effectiveness of this drug as a future partner in artemisinin-based combinations. No evidence of mutations linked to artemisinin resistance in Pfk13 was found, suggesting that parasites are sensitive to artemisinin derivatives in the study area. These findings are a baseline for routine molecular surveillance to proactively identify the emergence and spread of artemisinin-resistant parasites.

Distribution of Plasmodium falciparum K13 gene polymorphisms across transmission settings in Ghana.

Malaria is a significant global health concern, with a majority of cases in Sub-Saharan African nations. Numerous antimalarial drugs have been developed to counter the rampant prevalence of Plasmodium falciparum malaria. Artemisinin-based Combination Therapy (ACT) has served as the primary treatment of uncomplicated malaria in Ghana since 2005. However, a growing concern has emerged due to the escalating reports of ACT resistance, particularly in Southeast Asia, and its encroachment into Africa. Specifically, mutations in the Kelch propeller domain on chromosome 13 (Pfk13) have been linked to ACT resistance. Yet, our understanding of mutation prevalence in Africa remains largely uncharted. In this study, we compared Pfk13 sequences obtained from 172 P. falciparum samples across three ecological and transmission zones in Ghana. We identified 27 non-synonymous mutations among these sequences, of which two of the mutations, C580Y (found in two samples from the central region) and Y493H (found in one sample from the north), had previously been validated for their association with artemisinin resistance, a phenomenon widespread in Southeast Asia. The Pfk13 gene diversity was most pronounced in the northern savannah than the central forest and south coastal regions, where transmission rates are lower. The observed mutations were not significantly associated with geographical regions, suggesting a frequent spread of mutations across the country. The ongoing global surveillance of artemisinin resistance remains pivotal, and our findings provides insights into the potential spread of resistant parasites in West Africa. Furthermore, the identification of novel codon mutations in this study raises their potential association to ACT resistance, warranting further investigation through in vitro assays to ascertain their functional significance.

What exactly does the PfK13 C580Y mutation in Plasmodium falciparum influence?

BACKGROUND: The emergence and spread of artemisinin resistance threaten global malaria control and elimination goals, and encourage research on the mechanisms of drug resistance in malaria parasites. Mutations in Plasmodium falciparum Kelch 13 (PfK13) protein are associated with artemisinin resistance, but the unique or common mechanism which results in this resistance is unclear. METHODS: We analyzed the effects of the PfK13 mutation on the transcriptome and proteome of P. falciparum at different developmental stages. Additionally, the number of merozoites, hemozoin amount, and growth of P. falciparum 3D7C580Y and P. falciparum 3D7WT were compared. The impact of iron supplementation on the number of merozoites of P. falciparum 3D7C580Y was also examined. RESULTS: We found that the PfK13 mutation did not significantly change glycolysis, TCA, pentose phosphate pathway, or oxidative phosphorylation, but did reduce the expression of reproduction- and DNA synthesis-related genes. The reduced number of merozoites, decreased level of hemozoin, and slowed growth of P. falciparum 3D7C580Y were consistent with these changes. Furthermore, adding iron supply could increase the number of the merozoites of P. falciparum 3D7C580Y. CONCLUSIONS: These results revealed that the PfK13 mutation reduced hemoglobin ingestion, leading to artemisinin resistance, likely by decreasing the parasites' requirement for haem and iron. This study helps elucidate the mechanism of artemisinin resistance due to PfK13 mutations.

Dihydroartemisinin-piperaquine efficacy in Plasmodium falciparum treatment and prevalence of drug-resistant molecular markers along China-Myanmar border in 2014-2023.

OBJECTIVES: The study aims to monitor dihydroartemisinin-piperaquine (DHA-PPQ) efficacy in Plasmodium falciparum and detect molecular markers associated with its resistance. METHODS: The World Health Organization's standard protocol for therapeutic efficacy studies (TES) was performed from 2014 to 2018; integrated drug efficacy surveillance (iDES) was performed from from 2019 to July 2023. Molecular markers were detected by polymerase chain reaction. The association between gene mutations and delayed parasite clearance was analysed by multivariate logistic regression analysis. RESULTS: A total of 226 P. falciparum patients were enrolled in the TES from 2014 to 2018, and 26 patients with P. falciparum from Africa were recruited in the iDES from 2019 to July 2023. The PCR-adjusted clinical and parasitological cure rate was 93.7% (95% CI: 92.6-99.5%) in the TES and 96.2% (95% CI: 80.4-99.9%) in the iDEs. Twelve mutants and an overall 55.0% prevalence of pfK13 mutations were detected. Of them, G533S, C447R, C447S, N458Y, C469Y, and A676D were first detected out along the China-Myanmar border. Referred to the wild strain, adjusted odds ratios of treatment failure for G533S, N458Y, and P574L by 42 days were 7.54 (95% CI: 1.605-45.86), 13.68 (95% CI: 1.95-130.72), and 89.00 (95% CI: 1.98-2482.1), respectively. CONCLUSION: The efficacy of DHA-PPQ from 2014 to 2018 declined in comparison with 2003 to 2013, but it is still effective for treatment of P. falciparum malaria. Results of the iDES indicate a risk of artemisinin resistance in Africa. G533S, N458Y, and P574L are associated with delayed parasite clearance and treatment failure.

The many paths to artemisinin resistance in Plasmodium falciparum.

Emerging resistance against artemisinin (ART) poses a major challenge in controlling malaria. Parasites with mutations in PfKelch13, the major marker for ART resistance, are known to reduce hemoglobin endocytosis, induce unfolded protein response (UPR), elevate phosphatidylinositol-3-phosphate (PI3P) levels, and stimulate autophagy. Nonetheless, PfKelch13-independent resistance is also reported, indicating extensive complementation by reconfiguration in the parasite metabolome and transcriptome. These findings implicate that there may not be a single 'universal identifier' of ART resistance. This review sheds light on the molecular, transcriptional, and metabolic pathways associated with ART resistance, while also highlighting the interplay between cellular heterogeneity, environmental stress, and ART sensitivity.

Epidrugs as Promising Tools to Eliminate Plasmodium falciparum Artemisinin-Resistant and Quiescent Parasites.

The use of artemisinin and its derivatives has helped reduce the burden of malaria caused by Plasmodium falciparum. However, artemisinin-resistant parasites are able, in the presence of artemisinins, to stop their cell cycles. This quiescent state can alter the activity of artemisinin partner drugs leading to a secondary drug resistance and thus threatens malaria eradication strategies. Drugs targeting epigenetic mechanisms (namely epidrugs) are emerging as potential antimalarial drugs. Here, we set out to evaluate a selection of various epidrugs for their activity against quiescent parasites, to explore the possibility of using these compounds to counter artemisinin resistance. The 32 chosen epidrugs were first screened for their antiplasmodial activity and selectivity. We then demonstrated, thanks to the specific Quiescent-stage Survival Assay, that four epidrugs targeting both histone methylation or deacetylation as well as DNA methylation decrease the ability of artemisinin-resistant parasites to recover after artemisinin exposure. In the quest for novel antiplasmodial drugs with new modes of action, these results reinforce the therapeutic potential of epidrugs as antiplasmodial drugs especially in the context of artemisinin resistance.

The impact of anti-malarial markets on artemisinin resistance: perspectives from Burkina Faso.

BACKGROUND: Widespread artemisinin resistance in Africa could be catastrophic when drawing parallels with the failure of chloroquine in the 1970s and 1980s. This article explores the role of anti-malarial market characteristics in the emergence and spread of arteminisin resistance in African countries, drawing on perspectives from Burkina Faso. METHODS: Data were collected through in-depth interviews and focus group discussions. A representative sample of national policy makers, regulators, public and private sector wholesalers, retailers, clinicians, nurses, and community members were purposively sampled. Additional information was also sought via review of policy publications and grey literature on anti-malarial policies and deployment practices in Burkina Faso. RESULTS: Thirty seven in-depth interviews and 6 focus group discussions were conducted. The study reveals that the current operational mode of anti-malarial drug markets in Burkina Faso promotes arteminisin resistance emergence and spread. The factors are mainly related to the artemisinin-based combination therapy (ACT) supply chain, to ACT quality, ACT prescription monitoring and to ACT access and misuse by patients. CONCLUSION: Study findings highlight the urgent requirement to reform current characteristics of the anti-malarial drug market in order to delay the emergence and spread of artemisinin resistance in Burkina Faso. Four recommendations for public policy emerged during data analysis: (1) Address the suboptimal prescription of anti-malarial drugs, (2) Apply laws that prohibit the sale of anti-malarials without prescription, (3) Restrict the availability of street drugs, (4) Sensitize the population on the value of compliance regarding correct acquisition and intake of anti-malarials. Funding systems for anti-malarial drugs in terms of availability and accessibility must also be stabilized.

Plasmodium falciparum GCN5 plays a key role in regulating artemisinin resistance-related stress responses.

Plasmodium falciparum causes the most severe malaria and is exposed to various environmental and physiological stresses in the human host. Given that GCN5 plays a critical role in regulating stress responses in model organisms, we aimed to elucidate PfGCN5's function in stress responses in P. falciparum. The protein level of PfGCN5 was substantially induced under three stress conditions [heat shock, low glucose starvation, and dihydroartemisinin, the active metabolite of artemisinin (ART)]. With a TetR-DOZI conditional knockdown (KD) system, we successfully down-regulated PfGCN5 to ~50% and found that KD parasites became more sensitive to all three stress conditions. Transcriptomic analysis via RNA-seq identified ~1,000 up- and down-regulated genes in the wild-type (WT) and KD parasites under these stress conditions. Importantly, DHA induced transcriptional alteration of many genes involved in many aspects of stress responses, which were heavily shared among the altered genes under heat shock and low glucose conditions, including ART-resistance-related genes such as K13 and coronin. Based on the expression pattern between WT and KD parasites under three stress conditions, ~300-400 genes were identified to be involved in PfGCN5-dependent, general, and stress-condition-specific responses with high levels of overlaps among three stress conditions. Notably, using ring-stage survival assay, we found that KD or inhibition of PfGCN5 could sensitize the ART-resistant parasites to the DHA treatment. All these indicate that PfGCN5 is pivotal in regulating general and ART-resistance-related stress responses in malaria parasites, implicating PfGCN5 as a potential target for malaria intervention.

Artemisinin resistance in P. falciparum: probing the interacting partners of Kelch13 protein in parasite.

OBJECTIVES: Artemisinin (ART) resistance in Plasmodium is threatening the artemisinin combination therapies-the first line of defence against malaria. ART resistance has been established to be mediated by the Plasmodium Kelch13 (PfK13) protein. For the crucial role of PfK13 in multiple pathways of the Plasmodium life cycle and ART resistance, it is imperative that we investigate its interacting partners. METHODS: We recombinantly expressed PfK13-p (Bric a brac/Poxvirus and zinc finger and propeller domains), generating anti-PfK13-p antibodies to perform co-immunoprecipitation assays and probed PfK13 interacting partners. Surface plasmon resonance and pull-down assays were performed to establish physical interactions of representative proteins with PfK13-p. RESULTS: The co-immunoprecipitation assays identified 17 proteins with distinct functions in the parasite life cycle- protein folding, cellular metabolism, and protein binding and invasion. In addition to the overlap with previously identified proteins, our study identified 10 unique proteins. Fructose-biphosphate aldolase and heat shock protein 70 demonstrated strong biophysical interaction with PfK13-p, with KD values of 6.6 µM and 7.6 µM, respectively. Additionally, Plasmodium merozoite surface protein 1 formed a complex with PfK13-p, which is evident from the pull-down assay. CONCLUSION: This study adds to our knowledge of the PfK13 protein in mediating ART resistance by identifying new PfK13 interacting partners. Three representative proteins-fructose-biphosphate aldolase, heat shock protein 70, and merozoite surface protein 1-demonstrated clear evidence of biophysical interactions with PfK13-p. However, elucidation of the functional relevance of these physical interactions are crucial in context of PfK13 role in ART resistance.

Therapeutic efficacy of artesunate-amodiaquine and artemether-lumefantrine for the treatment of uncomplicated falciparum malaria in Chad: clinical and genetic surveillance.

BACKGROUND: Artesunate-amodiaquine (AS-AQ) and artemether-lumefantrine (AL) are the currently recommended first-and second-line therapies for uncomplicated Plasmodium falciparum infections in Chad. This study assessed the efficacy of these artemisinin-based combinations, proportion of day 3 positive patients, proportions of molecular markers associated with P. falciparum resistance to anti-malarial drugs and variable performance of HRP2-based malaria rapid diagnostic tests (RDTs). METHODS: A single-arm prospective study assessing the efficacy of AS-AQ and AL at three sites (Doba, Kelo and Koyom) was conducted between November 2020 to January 2021. Febrile children aged 6 to 59 months with confirmed uncomplicated P. falciparum infection were enrolled sequentially first to AS-AQ and then AL at each site and followed up for 28 days. The primary endpoint was PCR-adjusted adequate clinical and parasitological response (ACPR). Samples collected on day 0 were analysed for mutations in pfkelch13, pfcrt, pfmdr-1, pfdhfr, pfdhps genes and deletions in pfhrp2/pfhrp3 genes. RESULTS: By the end of 28-day follow-up, per-protocol PCR corrected ACPR of 97.8% (CI 95% 88.2-100) in Kelo and 100% in Doba and Kayoma were observed among AL treated patients. For ASAQ, 100% ACPR was found in all sites. All, but one patient, did not have parasites detected on day 3. Out of the 215 day 0 samples, 96.7% showed pfkelch13 wild type allele. Seven isolates carried nonsynonymous mutations not known to be associated artemisinin partial resistance (ART-R). Most of samples had a pfcrt wild type allele (79% to 89%). The most prevalent pfmdr-1 allele detected was the single mutant 184F (51.2%). For pfdhfr and pfdhps mutations, the quintuple mutant allele N51I/C59R/S108N + G437A/540E responsible for SP treatment failures in adults and children was not detected. Single deletion in the pfhrp2 and pfhrp3 gene were detected in 10/215 (4.7%) and 2/215 (0.9%), respectively. Dual pfhrp2/pfhrp3 deletions, potentially threatening the efficacy of HRP2-based RDTs, were observed in 5/215 (2.3%) isolates. CONCLUSION: The results of this study confirm that AS-AQ and AL treatments are highly efficacious in study areas in Chad. The absence of known pfkelch13 mutations in the study sites and the high parasite clearance rate at day 3 suggest the absence of ART-R. The absence of pfdhfr/pfdhps quintuple or sextuple (quintuple + 581G) mutant supports the continued use of SP for IPTp during pregnancy. The presence of parasites with dual pfhrp2/pfhrp3 deletions, potentially threatening the efficacy of HRP2-based RDTs, warrants the continued surveillance. Trial registration ACTRN12622001476729.

In Vitro Effect on Plasmodium falciparum and In Vivo Effect on Plasmodium berghei of Annomaal, an Oily Fraction Obtained from the Seeds of Annona squamosa.

Malaria remains a life-threatening health problem and is responsible for the high rates of mortality and morbidity in the tropical and subtropical regions of the world. The increasing threat of drug resistance to available artemisinin-based therapy warrants an urgent need to develop new antimalarial drugs that are safer, more effective, and have a novel mode of action. Natural plants are an excellent source of inspiration in searching for a new antimalarial agent. This research reports a systematic investigation for determining the antimalarial potential of the seeds of A. squamosa. The study shows that the crude seed extract (CSE), protein, saponin, and the oily fractions of the seeds were nontoxic at a 2000 mg/kg body weight dose when tested in Wistar rats, thus revealing high safety is classified as class 5. The oily fraction, Annomaal, demonstrated pronounced antimalarial activity with low IC50 (1.25 ± 0.183 µg/mL) against P. falciparum in vitro. The CSE and Annomaal significantly inhibited the growth of P. berghei parasites in vivo with 58.47% and 61.11% chemo suppression, respectively, while the standard drug artemether showed chemo suppression of 66.75%. Furthermore, the study demonstrated that oral administration of Annomaal at a daily dose of 250 mg/kg/day for 3 days was adequate to provide a complete cure to the P. berghei-infected mice. Annomaal thus holds promise as being patient-compliant due to the shorter treatment schedule, eliminating the need for frequent dosing for extended time periods as required by several synthetic antimalarial drugs. Further studies are needed to determine the active compounds in the oily fraction responsible for antimalarial activity.