Mutation in Plasmodium falciparum BTB/POZ domain of K13 protein confers artemisinin resistance

Partial artemisinin resistance, defined in patients as a delayed parasite clearance following artemisinin-based treatment, is conferred by non-synonymous mutations in the Kelch beta-propeller domain of the Plasmodium falciparum k13 ( pfk13 ) gene. Here, we carried out in vitro selection over a one-year period on a West African P. falciparum strain isolated from Kolle (Mali) under a dose-escalating artemisinin regimen. After 18 cycles of sequential drug pressure, the selected parasites exhibited enhanced survival to dihydroartemisinin in the ring-stage survival assay (RSA 0-3h = 9.2%). Sanger and whole-genome sequence analyses identified the PfK13 P413A mutation, localized in the BTB/POZ domain, upstream of the propeller domain. This mutation was sufficient to confer in vitro artemisinin resistance when introduced into the PfK13 coding sequence of the parasite strain Dd2 by CRISPR/Cas9 gene editing. These results together with structural studies of the protein demonstrate that the propeller domain is not the sole in vitro mediator of PfK13-mediated artemisinin resistance, and highlight the importance of monitoring for mutations throughout PfK13.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1813386115 ◽

2018 ◽

Vol 115 (49) ◽

pp. 12513-12518 ◽

Cited By ~ 29

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.

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Delayed Parasite Clearance after Treatment with Dihydroartemisinin-Piperaquine in Plasmodium falciparum Malaria Patients in Central Vietnam

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02746-14 ◽

2014 ◽

Vol 58 (12) ◽

pp. 7049-7055 ◽

Cited By ~ 50

Author(s):

Kamala Thriemer ◽

Nguyen Van Hong ◽

Anna Rosanas-Urgell ◽

Bui Quang Phuc ◽

Do Manh Ha ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Clearance Rate ◽

Artemisinin Resistance ◽

Parasite Clearance ◽

Clearance Time ◽

Sensitivity Testing ◽

Content Type ◽

Parasite Clearance Time ◽

Central Vietnam

ABSTRACTReduced susceptibility ofPlasmodium falciparumtoward artemisinin derivatives has been reported from the Thai-Cambodian and Thai-Myanmar borders. Following increasing reports from central Vietnam of delayed parasite clearance after treatment with dihydroartemisinin-piperaquine (DHA-PPQ), the current first-line treatment, we carried out a study on the efficacy of this treatment. Between September 2012 and February 2013, we conducted a 42-dayin vivoandin vitroefficacy study in Quang Nam Province. Treatment was directly observed, and blood samples were collected twice daily until parasite clearance. In addition, genotyping, quantitative PCR (qPCR), andin vitrosensitivity testing of isolates was performed. The primary endpoints were parasite clearance rate and time. The secondary endpoints included PCR-corrected and uncorrected cure rates, qPCR clearance profiles,in vitrosensitivity results (for chloroquine, dihydroartemisinin, and piperaquine), and genotyping for mutations in the Kelch 13 propeller domain. Out of 672 screened patients, 95 were recruited and 89 available for primary endpoint analyses. The median parasite clearance time (PCT) was 61.7 h (interquartile range [IQR], 47.6 to 83.2 h), and the median parasite clearance rate had a slope half-life of 6.2 h (IQR, 4.4 to 7.5 h). The PCR-corrected efficacy rates were estimated at 100% at day 28 and 97.7% (95% confidence interval, 91.2% to 99.4%) at day 42. At day 3, theP. falciparumprevalence by qPCR was 2.5 times higher than that by microscopy. The 50% inhibitory concentrations (IC50s) of isolates with delayed clearance times (≥72 h) were significantly higher than those with normal clearance times for all three drugs. Delayed parasite clearance (PCT, ≥72 h) was significantly higher among day 0 samples carrying the 543 mutant allele (47.8%) than those carrying the wild-type allele (1.8%;P= 0.048). In central Vietnam, the efficacy of DHA-PPQ is still satisfactory, but the parasite clearance time and rate are indicative of emerging artemisinin resistance. (This study has been registered at ClinicalTrials.gov under registration no. NCT01775592.)

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Plasmodium falciparum Founder Populations in Western Cambodia Have Reduced Artemisinin SensitivityIn Vitro

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03055-14 ◽

2014 ◽

Vol 58 (8) ◽

pp. 4935-4937 ◽

Cited By ~ 33

Author(s):

Chanaki Amaratunga ◽

Benoit Witkowski ◽

Dalin Dek ◽

Vorleak Try ◽

Nimol Khim ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Parasite Clearance ◽

Parasite Development ◽

Ring Stage ◽

Content Type ◽

Short Course ◽

Founder Populations ◽

Admixed Population ◽

Survival Assay

ABSTRACTReducedPlasmodium falciparumsensitivity to short-course artemisinin (ART) monotherapy manifests as a long parasite clearance half-life. We recently defined three parasite founder populations with long half-lives in Pursat, western Cambodia, where reduced ART sensitivity is prevalent. Using the ring-stage survival assay, we show that these founder populations have reduced ART sensitivityin vitroat the early ring stage of parasite development and that a genetically admixed population contains subsets of parasites with normal or reduced ART sensitivity.

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

10.1101/2021.01.05.425445 ◽

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.

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

10.21203/rs.2.24742/v2 ◽

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.

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

10.1101/2021.06.24.21259123 ◽

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.

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Identification of compounds active against quiescent artemisinin-resistant Plasmodium falciparum parasites via the quiescent-stage survival assay (QSA)

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkaa250 ◽

2020 ◽

Vol 75 (10) ◽

pp. 2826-2834

Author(s):

Thibaud Reyser ◽

Lucie Paloque ◽

Manel Ouji ◽

Michel Nguyen ◽

Sandie Ménard ◽

...

Keyword(s):

Artemisinin Resistance ◽

Laboratory Strain ◽

Parasite Clearance ◽

Quiescent State ◽

Field Isolates ◽

Pharmacological Targets ◽

Survival Assay ◽

Quiescent Stage ◽

Selection Of

Abstract Background Quiescence is an unconventional mechanism of Plasmodium survival, mediating artemisinin resistance. This phenomenon increases the risk of clinical failures following artemisinin-based combination therapies (ACTs) by slowing parasite clearance and allowing the selection of parasites resistant to partner drugs. Objectives To thwart this multiresistance, the quiescent state of artemisinin-resistant parasites must be taken into consideration from the very early stages of the drug discovery process. Methods We designed a novel phenotypic assay we have named the quiescent-stage survival assay (QSA) to assess the antiplasmodial activity of drugs on quiescent parasites. This assay was first validated on quiescent forms from different artemisinin-resistant parasite lines (laboratory strain and field isolates), using two reference drugs with different mechanisms of action: chloroquine and atovaquone. Furthermore, the efficacies of different partner drugs of artemisinins used in ACTs were investigated against both laboratory strains and field isolates from Cambodia. Results Our results highlight that because of the mechanism of quiescence and the respective pharmacological targets of drugs, drug efficacies on artemisinin-resistant parasites may be different between quiescent parasites and their proliferating forms. Conclusions These data confirm the high relevance of adding the chemosensitivity evaluation of quiescent parasites by the specific in vitro QSA to the antiplasmodial drug development process in the current worrisome context of artemisinin resistance.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01542-19 ◽

2019 ◽

Vol 64 (1) ◽

Cited By ~ 15

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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Artemisinin-Resistant Plasmodium falciparum Parasites Exhibit Altered Patterns of Development in Infected Erythrocytes

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00197-15 ◽

2015 ◽

Vol 59 (6) ◽

pp. 3156-3167 ◽

Cited By ~ 57

Author(s):

Amanda Hott ◽

Debora Casandra ◽

Kansas N. Sparks ◽

Lindsay C. Morton ◽

Geocel-Grace Castanares ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Artemisinin Resistance ◽

Drug Pressure ◽

Content Type ◽

Phenotypic Resistance ◽

Artemisinin Derivatives ◽

Stage Of Development ◽

Clinical Resistance

ABSTRACTArtemisinin derivatives are used in combination with other antimalarial drugs for treatment of multidrug-resistant malaria worldwide. Clinical resistance to artemisinin recently emerged in southeast Asia, yetin vitrophenotypes for discerning mechanism(s) of resistance remain elusive. Here, we describe novel phenotypic resistance traits expressed by artemisinin-resistantPlasmodium falciparum. The resistant parasites exhibit altered patterns of development that result in reduced exposure to drug at the most susceptible stage of development in erythrocytes (trophozoites) and increased exposure in the most resistant stage (rings). In addition, a novelin vitrodelayed clearance assay (DCA) that assesses drug effects on asexual stages was found to correlate with parasite clearance half-lifein vivoas well as with mutations in the Kelch domain gene associated with resistance (Pf3D7_1343700). Importantly, all of the resistance phenotypes were stable in cloned parasites for more than 2 years without drug pressure. The results demonstrate artemisinin-resistantP. falciparumhas evolved a novel mechanism of phenotypic resistance to artemisinin drugs linked to abnormal cell cycle regulation. These results offer insights into a novel mechanism of drug resistance inP. falciparumand new tools for monitoring the spread of artemisinin resistance.

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K13 Propeller Mutations in Plasmodium falciparum Populations in Regions of Malaria Endemicity in Vietnam from 2009 to 2016

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01578-16 ◽

2017 ◽

Vol 61 (4) ◽

Cited By ~ 23

Author(s):

Nguyen Thuy-Nhien ◽

Nguyen Kim Tuyen ◽

Nguyen Thanh Tong ◽

Nguyen Tuong Vy ◽

Ngo Viet Thanh ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Artemisinin Resistance ◽

Parasite Clearance ◽

Single Strain ◽

Content Type ◽

Reduced Ring ◽

Resistance Markers ◽

Multiple Strains

ABSTRACT The spread of artemisinin-resistant Plasmodium falciparum compromises the therapeutic efficacy of artemisinin combination therapies (ACTs) and is considered the greatest threat to current global initiatives to control and eliminate malaria. This is particularly relevant in Vietnam, where dihydroartemisinin-piperaquine (DP) is the recommended ACT for P. falciparum infection. The propeller domain gene of K13, a molecular marker of artemisinin resistance, was successfully sequenced in 1,060 P. falciparum isolates collected at 3 malaria hot spots in Vietnam between 2009 and 2016. Eight K13 propeller mutations (Thr474Ile, Tyr493His, Arg539Thr, Ile543Thr, Pro553Leu, Val568Gly, Pro574Leu, and Cys580Tyr), including several that have been validated to be artemisinin resistance markers, were found. The prevalences of K13 mutations were 29% (222/767), 6% (11/188), and 43% (45/105) in the Binh Phuoc, Ninh Thuan, and Gia Lai Provinces of Vietnam, respectively. Cys580Tyr became the dominant genotype in recent years, with 79.1% (34/43) of isolates in Binh Phuoc Province and 63% (17/27) of isolates in Gia Lai Province carrying this mutation. K13 mutations were associated with reduced ring-stage susceptibility to dihydroartemisinin (DHA) in vitro and prolonged parasite clearance in vivo. An analysis of haplotypes flanking K13 suggested the presence of multiple strains with the Cys580Tyr mutation rather than a single strain expanding across the three sites.

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