Discrete Survival Model Analysis of Plasmodium falciparum Response to Artemisinin-Based Combination Therapies among Children in Regions of Varying Malaria Transmission in Cameroon

The need to monitor changes in parasite clearance following treatment with artemisinin-based combination therapies (ACTs) is important in the containment of drug resistance. This study aimed to model Plasmodium falciparum response to ACTs among children in two different transmission settings (Mutengene and Garoua) in Cameroon. Using the step function, a discrete-time survival model was fitted with all the covariates included that might play a role in parasite clearance. The probability of clearing parasites within 24 h following treatment was 21.6% and 70.3% for younger children aged 6 to 59 months and 29.3% and 59.8% for older children aged 60 to 120 months in Mutengene and Garoua, respectively. After two days of treatment, the conditional probability of clearing parasites given that they were not cleared on day 1 was 76.7% and 96.6% for children aged 6–59 months and 83.1% and 93.5% for children aged 60–120 months in Mutengene and Garoua, respectively. The model demonstrated that the ecological setting, age group and pretreatment serum levels of creatinine and alanine aminotransferase were the main factors that significantly influenced parasite clearance in vivo after administration of ACTs (p < 0.05). The findings highlight the need for further investigations on host differential response to ACTs in current practice.

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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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Laboratory Detection of Artemisinin-Resistant Plasmodium falciparum

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01924-13 ◽

2014 ◽

Vol 58 (6) ◽

pp. 3157-3161 ◽

Cited By ~ 30

Author(s):

Kesinee Chotivanich ◽

Rupam Tripura ◽

Debashish Das ◽

Poravuth Yi ◽

Nicholas P. J. Day ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Parasite Clearance ◽

Ring Stage ◽

Clearance Time ◽

Content Type ◽

Reduced Ring ◽

Susceptibility Tests ◽

Low Sensitivity

ABSTRACTConventional 48-hin vitrosusceptibility tests have low sensitivity in identifying artemisinin-resistantPlasmodium falciparum, defined phenotypically by lowin vivoparasite clearance rates. We hypothesized originally that this discrepancy was explained by a loss of ring-stage susceptibility and so developed a simple field-adapted 24-h trophozoite maturation inhibition (TMI) assay focusing on the ring stage and compared it to the standard 48-h schizont maturation inhibition (WHO) test. In Pailin, western Cambodia, where artemisinin-resistantP. falciparumis prevalent, the TMI test mean (95% confidence interval) 50% inhibitory concentration (IC50) for artesunate was 6.8 (5.2 to 8.3) ng/ml compared with 1.5 (1.2 to 1.8) ng/ml for the standard 48-h WHO test (P= 0.001). TMI IC50s correlated significantly with thein vivoresponses to artesunate (parasite clearance time [r= 0.44,P= 0.001] and parasite clearance half-life [r= 0.46,P= 0.001]), whereas the standard 48-h test values did not. On continuous culture of two resistant isolates, the artemisinin-resistant phenotype was lost after 6 weeks (IC50s fell from 10 and 12 ng/ml to 2.7 and 3 ng/ml, respectively). Slow parasite clearance in falciparum malaria in western Cambodia results from reduced ring-stage susceptibility.

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Serious antimalaria resistance, genetic markers of Kelch 13, plasmepsine 2 CNV associated with dihydroartemisinine-piperaquine phosphate resistance in Plasmodium falciparum population in malaria hyperendemic zone of Dak Lak Province (2019-2020)

World Journal of Advanced Research and Reviews ◽

10.30574/wjarr.2020.8.1.0395 ◽

2020 ◽

Vol 8 (1) ◽

pp. 246-253

Author(s):

Huynh Hong Quang ◽

Chau Van Khanh ◽

Phạm Thanh Hien ◽

Nguyen Thanh Thuy Nhien ◽

Do Van Nguyen ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Molecular Markers ◽

Study Design ◽

Plasmodium Falciparum Malaria ◽

Parasite Clearance ◽

Controlled Study ◽

In Vivo Test ◽

Number Variation

Dihydroartemisinin-piperaquine (DHA-PPQ) is a current frontline drug recommended in global by WHO for the treatment of Plasmodium falciparum malaria (WHO, 2015), but is now failing in Vietnam’s provinces where border Cambodia, and has emerged and spread. The purpose of this study was to evaluate the efficacy and molecular markers of DHA-PPQ failures in Dak Lak province. Methods: A study design of non-randomized controlled study design for the 42 day-course follow-up in vivo test, and the molecular markers analysis. Findings: The data showed that adequate clinical and parasitological response was sharply declined to 12,1%, the proportion of late clinical failure was 51.5%, and 36.4% of patients had late parasitological. The proportion of positive parasitemia at D3 was 37%, the slope half-life was 5.36 hrs, and the progressive parasite clearance (PC) PC50, PC75, PC 90, PC95, and PC99 were 13.24; 19.29; 25.69; 29.97 and 39.15 hours, respectively. Molecular markers of C580Y Kelch mutation were observed in 100% (50/50) of the patients, the increased of Plasmepsine 2 copy number variation (CNV) was 72% (36/50), and the proportion of patients who had both K13 and increased Plasmepsine 2 CNV was 72% (36/50). Conclusions: The DHA-PPQ efficacy severely decreased to 12.1%, overall treatment failure was 87.9% with the prominent C580Y mutant plus increased Plasmepsine 2 CNV in delayed asexual P. falciparum parasite clearance. These obvious data suggest the urgency to change antimalarial policy in DHA-PPQ resistance zones, especially in Dak Lak province.

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Complex Polymorphisms in the Plasmodium falciparum Multidrug Resistance Protein 2 Gene and Its Contribution to Antimalarial Response

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03337-14 ◽

2014 ◽

Vol 58 (12) ◽

pp. 7390-7397 ◽

Cited By ~ 17

Author(s):

Maria Isabel Veiga ◽

Nuno S. Osório ◽

Pedro Eduardo Ferreira ◽

Oscar Franzén ◽

Sabina Dahlstrom ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Multidrug Resistance ◽

Abc Transporter ◽

Parasite Clearance ◽

Antimalarial Drugs ◽

Multidrug Resistance Protein ◽

Content Type ◽

Resistance Protein

ABSTRACTPlasmodium falciparumhas the capacity to escape the actions of essentially all antimalarial drugs. ATP-binding cassette (ABC) transporter proteins are known to cause multidrug resistance in a large range of organisms, including theApicomplexaparasites.P. falciparumgenome analysis has revealed two genes coding for the multidrug resistance protein (MRP) type of ABC transporters:Pfmrp1, previously associated with decreased parasite drug susceptibility, and the poorly studiedPfmrp2. The role ofPfmrp2polymorphisms in modulating sensitivity to antimalarial drugs has not been established. We herein report a comprehensive account of thePfmrp2genetic variability in 46 isolates from Thailand. A notably high frequency of 2.8 single nucleotide polymorphisms (SNPs)/kb was identified for this gene, including some novel SNPs. Additionally, we found thatPfmrp2harbors a significant number of microindels, some previously not reported. We also investigated the potential association of the identifiedPfmrp2polymorphisms with alteredin vitrosusceptibility to several antimalarials used in artemisinin-based combination therapy and with parasite clearance time. Association analysis suggestedPfmrp2polymorphisms modulate the parasite'sin vitroresponse to quinoline antimalarials, including chloroquine, piperaquine, and mefloquine, and association within vivoparasite clearance. In conclusion, our study reveals that thePfmrp2gene is the most diverse ABC transporter known inP. falciparumwith a potential role in antimalarial drug resistance.

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Little Polymorphism at the K13 Propeller Locus in Worldwide Plasmodium falciparum Populations Prior to the Introduction of Artemisinin Combination Therapies

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02370-15 ◽

2016 ◽

Vol 60 (6) ◽

pp. 3340-3347 ◽

Cited By ~ 17

Author(s):

Toshihiro Mita ◽

Richard Culleton ◽

Nobuyuki Takahashi ◽

Masatoshi Nakamura ◽

Takahiro Tsukahara ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Haplotype Diversity ◽

Artemisinin Resistance ◽

Pairwise Comparison ◽

Purifying Selection ◽

Parasite Clearance ◽

Combination Therapies ◽

Content Type ◽

Exposed Population ◽

Artemisinin Combination Therapies

The emergence and spread of artemisinin-resistantPlasmodium falciparumis of huge concern for the global effort toward malaria control and elimination. Artemisinin resistance, defined as a delayed time to parasite clearance following administration of artemisinin, is associated with mutations in thePfkelch13gene of resistant parasites. To date, as many as 60 nonsynonymous mutations have been identified in this gene, but whether these mutations have been selected by artemisinin usage or merely reflect natural polymorphism independent of selection is currently unknown. To clarify this, we sequenced thePfkelch13propeller domain in 581 isolates collected before (420 isolates) and after (161 isolates) the implementation of artemisinin combination therapies (ACTs), from various regions of endemicity worldwide. Nonsynonymous mutations were observed in 1% of parasites isolated prior to the introduction of ACTs. Frequencies of mutant isolates, nucleotide diversity, and haplotype diversity were significantly higher in the parasites isolated from populations exposed to artemisinin than in those from populations that had not been exposed to the drug. In the artemisinin-exposed population, a significant excess of dN compared to dS was observed, suggesting the presence of positive selection. In contrast, pairwise comparison of dN and dS and the McDonald and Kreitman test indicate that purifying selection acts on thePfkelch13propeller domain in populations not exposed to ACTs. These population genetic analyses reveal a low baseline ofPfkelch13polymorphism, probably due to purifying selection in the absence of artemisinin selection. In contrast, variousPfkelch13mutations have been selected under artemisinin pressure.

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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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Intrahost modeling of artemisinin resistance in Plasmodium falciparum

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1006113108 ◽

2010 ◽

Vol 108 (1) ◽

pp. 397-402 ◽

Cited By ~ 114

Author(s):

Sompob Saralamba ◽

Wirichada Pan-Ngum ◽

Richard J. Maude ◽

Sue J. Lee ◽

Joel Tarning ◽

...

Keyword(s):

Mathematical Model ◽

Plasmodium Falciparum ◽

Goodness Of Fit ◽

Artemisinin Resistance ◽

Parasite Clearance ◽

Model Parameters ◽

Asexual Parasite ◽

Ring Stage ◽

Reduced Ring

Artemisinin-resistant Plasmodium falciparum malaria has emerged in western Cambodia. Resistance is characterized by prolonged in vivo parasite clearance times (PCTs) following artesunate treatment. The biological basis is unclear. The hypothesis that delayed parasite clearance results from a stage-specific reduction in artemisinin sensitivity of the circulating young asexual parasite ring stages was examined. A mathematical model was developed, describing the intrahost parasite stage-specific pharmacokinetic–pharmacodynamic relationships. Model parameters were estimated using detailed pharmacokinetic and parasite clearance data from 39 patients with uncomplicated falciparum malaria treated with artesunate from Pailin (western Cambodia) where artemisinin resistance was evident and 40 patients from Wang Pha (northwestern Thailand) where efficacy was preserved. The mathematical model reproduced the observed parasite clearance for each patient with an accurate goodness of fit (rmsd: 0.03–0.67 in log10 scale). The parameter sets that provided the best fits with the observed in vivo data consist of a highly conserved concentration–effect relationship for the trophozoite and schizont parasite stages, but a variable relationship for the ring stages. The model-derived assessment suggests that the efficacy of artesunate on ring stage parasites is reduced significantly in Pailin. This result supports the hypothesis that artemisinin resistance mainly reflects reduced ring-stage susceptibility and predicts that doubling the frequency of dosing will accelerate clearance of artemisinin-resistant parasites.

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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 Mutations in Antimalarial Resistance Gene Kelch13 from Plasmodium falciparum Isolates in Kano, Nigeria

Tropical Medicine and Infectious Disease ◽

10.3390/tropicalmed5020085 ◽

2020 ◽

Vol 5 (2) ◽

pp. 85

Author(s):

Umar F. Abubakar ◽

Ruqayya Adam ◽

Muhammad M. Mukhtar ◽

Abdullahi Muhammad ◽

Adamu A. Yahuza ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Potential Role ◽

Parasite Clearance ◽

Falciparum Infection ◽

Combination Therapies ◽

First Line ◽

High Prevalence ◽

Antimalarial Resistance ◽

Artemisinin Combination Therapies

Malaria control relies on first-line treatments that use artemisinin-combination therapies (ACT). Unfortunately, mutations in the plasmodium falciparum kelch13 gene result in delayed parasite clearance. Research on what is causing ACT failure is non-existent in northwestern Nigeria. Thus, the presence of mutations in kelch13 in P. falciparum isolates from Kano, Nigeria was investigated in this study. Microscopic examination of 154 blood samples obtained from patients revealed a high prevalence of P. falciparum infection (114 positive individuals, slide positivity rate = 74.03%). The 114 patients were administered Cartef® (ACT) and out of the 50 patients that returned for the 14-day follow up, 11 were positive for P. falciparum (slide positivity rate = 22%). On day 0, 80 samples out of 114 and 11 samples on day 14 (91 out of 125 microscopy-positive samples) were positive with Plasmodium according to the PCR of cytochrome oxidase I, which corresponds to 72.8%. A fragment of the kelch13 gene encompassing the propeller domains was sequenced in 49 samples, alongside samples of the susceptible strain pf_3D7. Low polymorphism was observed, suggesting a lack of selection on this gene, and only six mutations (Glu433Gly, Phe434Ile, Phe434Ser, Ile684Asn, Ile684Thr and Glu688Lys) were found. The epidemiologic impact of these mutations and their potential role in ACT resistance needs to be investigated further.

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A comparison of the in vivo kinetics of Plasmodium falciparum ring–infected erythrocyte surface antigen–positive and –negative erythrocytes

Blood ◽

10.1182/blood.v98.2.450 ◽

2001 ◽

Vol 98 (2) ◽

pp. 450-457 ◽

Cited By ~ 46

Author(s):

Paul N. Newton ◽

Kesinee Chotivanich ◽

Wirongrong Chierakul ◽

Ronatrai Ruangveerayuth ◽

Pramote Teerapong ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Surface Antigen ◽

Infected Erythrocyte ◽

Parasite Clearance ◽

Median Percentage ◽

Cell Life ◽

Erythrocyte Surface ◽

The Mean ◽

Kinetics Of

Ring-infected erythrocyte surface antigen (RESA)-positive,Plasmodium falciparum–negative red blood cells (RBCs) are cells from which the malaria parasite has been removed by the host without the destruction of the erythrocyte (“pitting”). The survival of RESA-RBCs in vivo was assessed in 14 severe and 6 uncomplicated falciparum malaria patients. The mean RESA-RBC life of 183 hours (95% confidence interval [CI], 136-246) was longer than the median parasite clearance time of 66 hours (range, 30-108 hours) but shorter than the mean red cell life of 1027 hours (95% CI, 840-1213) (P = .0004), with a median ratio of 0.2:1.0 (range, 0.1-0.7). The estimated median percentage of parasites pitted/body transit was 0.003% (range, 0.001%-0.05%). The rate of rise of the RESA-RBC count during the first 24 hours after antimalarial treatment was significantly faster (P = .036) and the subsequent RESA-RBC survival significantly shorter (P = .017) after treatment with an artemisinin derivative than after treatment with quinine. Parasitization of red cells leads to changes in the erythrocyte that shorten their survival even if the parasite is removed subsequently.

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