scholarly journals The extended recovery ring-stage survival assay provides superior prediction of patient clearance half-life and increases throughput

2019 ◽  
Author(s):  
Sage Z. Davis ◽  
Puspendra P. Singh ◽  
Katelyn M. Vendrely ◽  
Douglas A. Shoue ◽  
Lisa A. Checkley ◽  
...  
Keyword(s):  
Southeast Asia ◽  
Half Life ◽  
Artemisinin Resistance ◽  
Pcr Amplification ◽  
Fold Change ◽  
Ring Stage ◽  
Post Exposure ◽  
Survival Assay

Abstract Background Tracking and understanding artemisinin resistance is key for preventing global setbacks in malaria eradication efforts. The ring-stage survival assay (RSA) is the current gold standard for in vitro artemisinin resistance phenotyping. However, the RSA has several drawbacks: it is relatively low throughput, has high variance due to microscopy readout, and correlates poorly with the current benchmark for in vivo resistance, patient clearance half-life post-artemisinin treatment. Here a modified RSA is presented, the extended Recovery Ring-stage Survival Assay (eRRSA), using 15 cloned patient isolates from Southeast Asia with a range of patient clearance half-lives, including parasite isolates with and without kelch13 mutations. Methods P. falciparum cultures were synchronized with single layer Percoll during the schizont stage of the erythrocytic cycle. Cultures were left to reinvade to early ring-stage and parasitemia was quantified using flow cytometry. Cultures were diluted to 2% hematocrit and 0.5% parasitemia in a 96-well plate to start the assay, allowing for increased throughput and decreased variability between biological replicates. Parasites were treated with 700nM of dihydroartemisinin or an equivalent amount of dimethyl sulfoxide (DMSO) for 6 h, washed three times in drug-free media, and incubated for 66 or 114 h, when samples were collected and frozen for PCR amplification. A SYBR Green-based quantitative PCR method was used to quantify the fold-change between treated and untreated samples. Results 15 cloned patient isolates from Southeast Asia with a range of patient clearance half-lives were assayed using the eRRSA. Due to the large number of pyknotic and dying parasites at 66 h post-exposure (72 h sample), parasites were grown for an additional cell cycle (114 h post-exposure, 120 h sample), which drastically improved correlation with patient clearance half-life compared to the 66 h post-exposure sample. A Spearman correlation of 0.8393 between fold change and patient clearance half-life was identified in these 15 isolates from Southeast Asia, which is the strongest correlation reported to date. Conclusions eRRSA drastically increases the efficiency and accuracy of in vitro artemisinin resistance phenotyping compared to the traditional RSA, which paves the way for extensive in vitro phenotyping of hundreds of artemisinin resistant parasites.

scholarly journals The extended recovery ring-stage survival assay provides a superior association with patient clearance half-life and increases throughput

2020 ◽  
Author(s):  
Sage Z. Davis ◽  
Puspendra P. Singh ◽  
Katelyn M. Vendrely ◽  
Douglas A. Shoue ◽  
Lisa A. Checkley ◽  
...  
Keyword(s):  
Southeast Asia ◽  
Half Life ◽  
Artemisinin Resistance ◽  
Pcr Amplification ◽  
Fold Change ◽  
Ring Stage ◽  
Post Exposure ◽  
Survival Assay

Abstract Background Tracking and understanding artemisinin resistance is key for preventing global setbacks in malaria eradication efforts. The ring-stage survival assay (RSA) is the current gold standard for in vitro artemisinin resistance phenotyping. However, the RSA has several drawbacks: it is relatively low throughput, has high variance due to microscopy readout, and correlates poorly with the current benchmark for in vivo resistance, patient clearance half-life post-artemisinin treatment. Here a modified RSA is presented, the extended Recovery Ring-stage Survival Assay (eRRSA), using 15 cloned patient isolates from Southeast Asia with a range of patient clearance half-lives, including parasite isolates with and without kelch13 mutations. Methods P. falciparum cultures were synchronized with single layer Percoll during the schizont stage of the erythrocytic cycle. Cultures were left to reinvade to early ring-stage and parasitemia was quantified using flow cytometry. Cultures were diluted to 2% hematocrit and 0.5% parasitemia in a 96-well plate to start the assay, allowing for increased throughput and decreased variability between biological replicates. Parasites were treated with 700nM of dihydroartemisinin or an equivalent amount of dimethyl sulfoxide (DMSO) for 6 h, washed three times in drug-free media, and incubated for 66 or 114 h, when samples were collected and frozen for PCR amplification. A SYBR Green-based quantitative PCR method was used to quantify the fold-change between treated and untreated samples. Results 15 cloned patient isolates from Southeast Asia with a range of patient clearance half-lives were assayed using the eRRSA. Due to the large number of pyknotic and dying parasites at 66 h post-exposure (72 h sample), parasites were grown for an additional cell cycle (114 h post-exposure, 120 h sample), which drastically improved correlation with patient clearance half-life compared to the 66 h post-exposure sample. A Spearman correlation of 0.8393 between fold change and patient clearance half-life was identified in these 15 isolates from Southeast Asia, which is the strongest correlation reported to date. Conclusions eRRSA drastically increases the efficiency and accuracy of in vitro artemisinin resistance phenotyping compared to the traditional RSA, which paves the way for extensive in vitro phenotyping of hundreds of artemisinin resistant parasites.

scholarly journals The extended recovery ring stage survival assay provides superior prediction of patient clearance half life and increases throughput

2019 ◽  
Author(s):  
Sage Z. Davis ◽  
Puspendra P. Singh ◽  
Katelyn M. Vendrely ◽  
Douglas A. Shoue ◽  
Lisa A. Checkley ◽  
...  
Keyword(s):  
Southeast Asia ◽  
Half Life ◽  
Artemisinin Resistance ◽  
Pcr Amplification ◽  
Fold Change ◽  
Ring Stage ◽  
Post Exposure ◽  
Survival Assay

AbstractBackgroundTracking and understanding artemisinin resistance is key for preventing global setbacks in malaria eradication efforts. The ring-stage survival assay (RSA) is the current gold standard for in vitro artemisinin resistance phenotyping. However, the RSA has several drawbacks: it is relatively low throughput, has high variance due to microscopy readout, and correlates poorly with the current benchmark for in vivo resistance, patient clearance half-life post-artemisinin treatment. Here a modified RSA is presented, the extended Recovery Ring-stage Survival Assay (eRRSA), using 15 cloned patient isolates from Southeast Asia with a range of patient clearance half-lives, including parasite isolates with and without kelch13 mutations.MethodsP. falciparum cultures were synchronized with single layer Percoll during the schizont stage of the erythrocytic cycle. Cultures were left to reinvade to early ring-stage and parasitemia was quantified using flow cytometry. Cultures were diluted to 2% hematocrit and 0.5% parasitemia in a 96-well plate to start the assay, allowing for increased throughput and decreased variability between biological replicates. Parasites were treated with 700nM of dihydroartemisinin or an equivalent amount of dimethyl sulfoxide (DMSO) for 6 h, washed three times in drug-free media, and incubated for 66 or 114 h, when samples were collected and frozen for PCR amplification. A SYBR Green-based quantitative PCR method was used to quantify the fold-change between treated and untreated samples.Results15 cloned patient isolates from Southeast Asia with a range of patient clearance half-lives were assayed using the eRRSA. Due to the large number of pyknotic and dying parasites at 66 h post-exposure (72 h sample), parasites were grown for an additional cell cycle (114 h post-exposure, 120 h sample), which drastically improved correlation with patient clearance half-life compared to the 66 h post-exposure sample. A Spearman correlation of 0.8393 between fold change and patient clearance half-life was identified in these 15 isolates from Southeast Asia, which is the strongest correlation reported to date.ConclusionseRRSA drastically increases the efficiency and accuracy of in vitro artemisinin resistance phenotyping compared to the traditional RSA, which paves the way for extensive in vitro phenotyping of hundreds of artemisinin resistant parasites.

scholarly journals Plasmodium falciparum K13 mutations in Africa and Asia present varying degrees of artemisinin resistance and an elevated fitness cost in African parasites

2021 ◽  
Author(s):  
Barbara H. Stokes ◽  
Kelly Rubiano ◽  
Satish K. Dhingra ◽  
Sachel Mok ◽  
Judith Straimer ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Southeast Asia ◽  
Survival Rates ◽  
Point Mutations ◽  
Artemisinin Resistance ◽  
Parasite Clearance ◽  
Definitive Evidence ◽  
The Impact

AbstractThe emergence of artemisinin (ART) resistance in Plasmodium falciparum parasites has led to increasing rates of treatment failure with first-line ART-based combination therapies (ACTs) in Southeast Asia. In this region, select mutations in K13 can result in delayed parasite clearance rates in vivo and enhanced survival in the ring-stage survival assay (RSA) in vitro. Our genotyping of 3,299 P. falciparum isolates across 11 sub-Saharan countries reveals the continuing dominance of wild-type K13 and confirms the emergence of a K13 R561H variant in Rwanda. Using gene editing, we provide definitive evidence that this mutation, along with M579I and C580Y, can confer variable degrees of in vitro ART resistance in African P. falciparum strains. C580Y and M579I were both associated with substantial fitness costs in African parasites, which may counter-select against their dissemination in high-transmission settings. We also report the impact of multiple K13 mutations, including the predominant variant C580Y, on RSA survival rates and fitness in multiple Southeast Asian strains. No change in ART susceptibility was observed upon editing point mutations in ferrodoxin or mdr2, earlier associated with ART resistance in Southeast Asia. These data point to the lack of an evident biological barrier to mutant K13 mediating ART resistance in Africa, while identifying their detrimental impact on parasite growth.

scholarly journals Modification of an atypical clathrin-independent AP-2 adaptin complex of Plasmodium falciparum reduces susceptibility to artemisinin

2019 ◽  
Author(s):  
Ryan C. Henrici ◽  
Rachel L. Edwards ◽  
Martin Zoltner ◽  
Donelly A. van Schalkwyk ◽  
Melissa N. Hart ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Artemisinin Resistance ◽  
Conditional Knockout ◽  
Parental Line ◽  
Ring Stage ◽  
Intracellular Traffic ◽  
Reduced Ring ◽  
Kelch Domain

SummaryThe efficacy of current antimalarial drugs is threatened by reduced susceptibility of Plasmodium falciparum to artemisinin. In the Mekong region this is associated with mutations in the kelch propeller-encoding domain of pfkelch13, but variants of other parasite proteins are also thought to modulate the response to drug. Evidence from human and rodent studies suggests that the μ-subunit of the AP-2 adaptin trafficking complex is one such protein of interest. We generated transgenic Plasmodium falciparum parasites encoding the I592T variant of pfap2μ, orthologous to the I568T mutation associated with in vivo artemisinin resistance in P. chabaudi. When exposed to a four-hour pulse of dihydroartemisin in the ring-stage survival assay, two P. falciparum clones expressing AP-2μ I592T displayed significant and reproducible survival of 8.0% and 10.3%, respectively, compared to <2% for the 3D7 parental line (P = 0.0011 for each clone). In immunoprecipitation and localisation studies of HA-tagged AP-2μ, we identified interacting partners including AP-2α, AP-1/2β, AP-2σ and a kelch-domain protein encoded on chromosome 10 of P. falciparum, K10. Conditional knockout indicates that the AP-2 trafficking complex in P. falciparum is essential for the fidelity of merozoite biogenesis and membrane organisation in the mature schizont. We also show that while other heterotetrameric AP-complexes and secretory factors interact with clathrin, AP-2 complex subunits do not. Thus, the AP-2 complex may be diverted from a clathrin-dependent endocytic role seen in most eukaryotes into a Plasmodium-specific function. These findings represent striking divergences from eukaryotic dogma and support a role for intracellular traffic in determining artemisinin sensitivity in vitro, confirming the existence of multiple functional routes to reduced ring-stage artemisinin susceptibility. Therefore, the utility of pfkelch13 variants as resistance markers is unlikely to be universal, and phenotypic surveillance of parasite susceptibility in vivo may be needed to identify threats to our current combination therapies.

scholarly journals Plasmodium berghei K13 Mutations Mediate In Vivo Artemisinin Resistance That is Reversed by Proteasome Inhibition

2020 ◽  
Author(s):  
Nelson V. Simwela ◽  
Barbara H. Stokes ◽  
Dana Aghabi ◽  
Matt Bogyo ◽  
David A. Fidock ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Gene Editing ◽  
Artemisinin Resistance ◽  
Causal Role ◽  
Ring Stage ◽  
Parasite Resistance ◽  
Mutant Lines

ABSTRACTThe recent emergence of Plasmodium falciparum (PF) parasite resistance to the first line antimalarial drug artemisinin is of particular concern. Artemisinin resistance is primarily driven by mutations in the PF K13 protein, which enhance survival of early ring stage parasites treated with the artemisinin active metabolite dihydroartemisinin in vitro and associate with delayed parasite clearance in vivo. However, association of K13 mutations with in vivo artemisinin resistance has been problematic due to the absence of a tractable model. Herein, we have employed CRISPR/Cas9 genome editing to engineer selected orthologous PF K13 mutations into the K13 gene of an artemisinin-sensitive, P. berghei (PB) rodent model of malaria. Introduction of the orthologous PF K13 F446I, M476I, Y493H and R539T mutations into PB K13 produced gene-edited parasites with reduced susceptibility to dihydroartemisinin in the standard 24-hour in vitro assay and increased survival in an adapted in vitro ring-stage survival assay. Mutant PB K13 parasites also displayed delayed clearance in vivo upon treatment with artesunate and achieved faster recrudescence upon treatment with artemisinin. Orthologous C580Y and I543T mutations could not be introduced into PB while the equivalent of the M476I and R539T mutations resulted in significant growth defects. Furthermore, a Plasmodium-selective proteasome inhibitor strongly synergized dihydroartemisinin action in these PB K13 mutant lines, providing further evidence that the proteasome can be targeted to overcome ART resistance. Taken together, our work provides clear experimental evidence for the involvement of K13 polymorphisms in mediating susceptibility to artemisinins in vitro, and most importantly under in vivo conditions.IMPORTANCERecent successes in malaria control have been seriously threatened by the emergence of Plasmodium falciparum parasite resistance to the frontline artemisinin drugs in Southeast Asia. P. falciparum artemisinin resistance is associated with mutations in the parasite K13 protein, which associates with a delay in the time required to clear the parasites upon treatment with the drug. Gene editing technologies have been used to validate the role of several candidate K13 mutations in mediating P. falciparum artemisinin resistance in vitro under laboratory conditions. Nonetheless, the causal role of these mutations under in vivo conditions has been a matter of debate. Here, we have used CRISPR/Cas9 gene editing to introduce K13 mutations associated with artemisinin resistance into the related rodent-infecting parasite, P. berghei. Phenotyping of these P. berghei K13 mutant parasites provides evidence of their role in mediating artemisinin resistance in vivo, which supports in vitro artemisinin resistance observations. However, we were unable to introduce some of the P. falciparum K13 mutations (C580Y, I543T) into the corresponding amino acid residues, while other introduced mutations (M476I, R539T equivalents) carried a pronounced fitness cost. Our study provides evidence of a clear causal role of K13 mutations in modulating susceptibility to artemisinins in vitro and in vivo using the well-characterized P. berghei model. We also show that inhibition of the P. berghei proteasome offsets parasite resistance to artemisinins in these mutant lines.

scholarly journals Artemisinin resistance phenotypes and K13 inheritance in a Plasmodium falciparum cross and Aotus model

2018 ◽  
Vol 115 (49) ◽  
pp. 12513-12518 ◽  
Author(s):  
Juliana M. Sá ◽  
Sarah R. Kaslow ◽  
Michael A. Krause ◽  
Viviana A. Melendez-Muniz ◽  
Rebecca E. Salzman ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Artemisinin Resistance ◽  
Parasite Clearance ◽  
Wild Type ◽  
Infected Monkey ◽  
Allelic Substitution ◽  
Survival Assay ◽  
Mean Differences

Concerns about malaria parasite resistance to treatment with artemisinin drugs (ARTs) have grown with findings of prolonged parasite clearance t1/2s (>5 h) and their association with mutations in Plasmodium falciparum Kelch-propeller protein K13. Here, we describe a P. falciparum laboratory cross of K13 C580Y mutant with C580 wild-type parasites to investigate ART response phenotypes in vitro and in vivo. After genotyping >400 isolated progeny, we evaluated 20 recombinants in vitro: IC50 measurements of dihydroartemisinin were at similar low nanomolar levels for C580Y- and C580-type progeny (mean ratio, 1.00; 95% CI, 0.62–1.61), whereas, in a ring-stage survival assay, the C580Y-type progeny had 19.6-fold (95% CI, 9.76–39.2) higher average counts. In splenectomized Aotus monkeys treated with three daily doses of i.v. artesunate, t1/2 calculations by three different methods yielded mean differences of 0.01 h (95% CI, −3.66 to 3.67), 0.80 h (95% CI, −0.92 to 2.53), and 2.07 h (95% CI, 0.77–3.36) between C580Y and C580 infections. Incidences of recrudescence were 57% in C580Y (4 of 7) versus 70% in C580 (7 of 10) infections (−13% difference; 95% CI, −58% to 35%). Allelic substitution of C580 in a C580Y-containing progeny clone (76H10) yielded a transformant (76H10C580Rev) that, in an infected monkey, recrudesced regularly 13 times over 500 d. Frequent recrudescences of ART-treated P. falciparum infections occur with or without K13 mutations and emphasize the need for improved partner drugs to effectively eliminate the parasites that persist through the ART component of combination therapy.

scholarly journals Phenotypic Changes in Artemisinin-Resistant Plasmodium falciparum LinesIn Vitro: Evidence for Decreased Sensitivity to Dormancy and Growth Inhibition

2011 ◽  
Vol 56 (1) ◽  
pp. 428-431 ◽  
Author(s):  
Franka Teuscher ◽  
Nanhua Chen ◽  
Dennis E. Kyle ◽  
Michelle L. Gatton ◽  
Qin Cheng
Keyword(s):  
Plasmodium Falciparum ◽  
Growth Arrest ◽  
Artemisinin Resistance ◽  
Mature Stage ◽  
Ring Stage ◽  
Content Type ◽  
Resistance Phenotype ◽  
Temporary Growth

ABSTRACTThe appearance ofPlasmodium falciparumparasites with decreasedin vivosensitivity but no measurablein vitroresistance to artemisinin has raised the urgent need to characterize the artemisinin resistance phenotype. Changes in the temporary growth arrest (dormancy) profile of parasites may be one aspect of this phenotype. In this study, we investigated the link between dormancy and resistance, using artelinic acid (AL)-resistant parasites. Our results demonstrate that the AL resistance phenotype has (i) decreased sensitivity of mature-stage parasites, (ii) decreased sensitivity of the ring stage to the induction of dormancy, and (iii) a faster recovery from dormancy.

scholarly journals Molecular assessment of kelch13 non-synonymous mutations in Plasmodium falciparum isolates from Central African Republic (2017–2019)

2020 ◽  
Author(s):  
Romaric Nzoumbou-Boko ◽  
Chris-Boris Gildas Panté-Wockama ◽  
Carine Ngoagoni ◽  
Nathalie Petiot ◽  
Eric Legrand ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Southeast Asia ◽  
Central African Republic ◽  
Artemisinin Resistance ◽  
Plasmodium Species ◽  
Sub Saharan Africa ◽  
Artemisinin Derivatives ◽  
Synonymous Mutations

Abstract Background: Over the last decade, Artemisinin-based Combination Therapies (ACT) have contributed substantially to the decrease in malaria-related morbidity and mortality. The emergence of Plasmodium falciparum parasites resistant to artemisinin derivatives in Southeast Asia and the risk of their spread or of local emergence in sub-Saharan Africa are a major threat to public health. This study thus set out to estimate the proportion of P. falciparum isolates, with PfKelch13 gene mutations associated with artemisinin resistance previously detected in Southeast Asia. Methods: Blood samples were collected in two sites of Bangui, the capital of the Central African Republic form 2017 to 2019. DNA was extracted and nested PCR were carried out to detect Plasmodium species and mutations in the propeller domain of the PfKelch13 gene. Results: A total of 255 P. falciparum isolates were analyzed. Among them, P. ovale DNA was found in four samples (1.57%, 4/255). Of 187 samples with interpretable PfKelch13 sequences, four isolates presented a mutation in the PfKelch13 gene (2.1%, 4/187), including one non-synonymous mutation (Y653N) (0.5%, 1/187). This mutation has never been described as associated with artemisinin resistance in Southeast Asia and its in vitro phenotype is unknown. Conclusion: This preliminary study indicates the need for a larger study on samples collected across the whole country along with the evaluation of in vitro and in vivo phenotype profiles of PfKelch13 mutant parasites to estimate the risk of artemisinin resistance in the CAR.

scholarly journals In vivo delayed clearance of Plasmodium falciparum malaria independent of kelch13 polymorphisms and with escalating malaria in Bangladesh.

2021 ◽  
Author(s):  
Maisha Khair Nima ◽  
Saiful Arefeen Sazed ◽  
Angana Mukherjee ◽  
Muhammad Riadul Haque Hossainey ◽  
Ching Swe Phru ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Half Life ◽  
Artemisinin Combination Therapy ◽  
Artemisinin Resistance ◽  
Plasmodium Falciparum Malaria ◽  
Parasite Clearance ◽  
Resistance Mutations ◽  
Emergence Of Resistance

The emergence of resistance to artemisinin drugs threatens global malaria control. Resistance is widely seen in South East Asia (SEA) and Myanmar, but not comprehensively assessed in Bangladesh. This is due to lack of measuring parasite clearance times in response to drug treatment, a gold standard used to track artemisinin resistance (AR), in the Chittagong Hill Tracts (CHT), where >90% of malaria occurs in Bangladesh. Here we report delay in clinical parasite clearance half-lives > 5 h characteristic of AR, in Bandarban, a south–eastern rural, CHT district with escalating malaria and bordering Myanmar. Thirty–one and 68 malaria patients respectively presented in the clinic in 2018 and 2019, and this increase well correlated to the district–level malaria surge and rise in rainfall, humidity and temperature. A total of 27 patients with uncomplicated Plasmodium falciparum malaria mono–infection, after administration of an artemisinin combination therapy (ACT) showed median (range) parasite clearance half–life and time of 5.6 (1.5 —9.6) and 24 (12—48) hours (h) respectively. The frequency distribution of parasite clearance half–life and time was bimodal, with a slower parasite clearance of 8 h in 20% of the participants. There was however, no detectable parasitemia 72 h after initiating ACT. Half-life clearance of > 5h, respectively seen in 35% and 40% of participants in 2018 and 2019, lacked in correlation to initial parasitemia, blood count parameters or resistance mutations of PfKelch13 (K13, the major parasite marker of AR). Culture adapted strains await assessment of in vitro resistance and new parasite determinants of AR.

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

2019 ◽  
Vol 64 (1) ◽  
Author(s):  
Ryan C. Henrici ◽  
Donelly A. van Schalkwyk ◽  
Colin J. Sutherland
Keyword(s):  
Plasmodium Falciparum ◽  
Artemisinin Resistance ◽  
Antimalarial Drugs ◽  
Ring Stage ◽  
Content Type ◽  
Adaptive Mechanisms ◽  
Transgenic Parasites ◽  
Survival Assay

ABSTRACT 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.

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