Artemisinin combination therapy fails even in the absence of Plasmodium falciparum kelch13 gene polymorphism in Central India

AbstractArtemisinin is the frontline fast-acting anti-malarial against P. falciparum. Emergence and spread of resistant parasite in eastern-India poses a threat to national malaria control programs. Therefore, the objective of our study is to evaluate the artesunate-sulfadoxine-pyrimethamine efficacy in Central India. 180 monoclonal P. falciparum-infected patients received standard ASSP therapy during August 2015–January 2017, soon after diagnosis and monitored over next 42-days. Artemisinin-resistance was assessed through in-vivo parasite clearance half-life (PC1/2), ex-vivo ring-stage survivability (RSA), and genome analysis of kelch13 and other candidate gene (pfcrt, pfmdr1, pfatpase 6, pfdhfr and pfdhps). Of 180 P. falciparum positive patients, 9.5% showed increased PC1/2 (> 5.5 h), among them eleven isolates (6.1%) showed reduced sensitivity to RSA. In 4.4% of cases, parasites were not cleared by 72 h and showed prolonged PC1/2(5.6 h) (P < 0.005) along with significantly higher RSA (2.2%) than cured patients (0.4%). None of day-3 positive isolates contained the pfkelch13 mutation implicated in artemisinin resistance. Parasite recrudescence was observed in 5.6% patients, which was associated with triple dhfr–dhps (A16I51R59N108I164–S436G437K540G581T613) combination mutation. Emergence of reduced sensitivity to artesunate-sulfadoxine-pyrimethamine, in central India highlighted the risk toward spread of resistant parasite across different parts of India. Day-3 positive parasite, featuring the phenotype of artemisinin-resistance without pfkelch13 mutation, suggested kelch13-independent artemisinin-resistance.

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Novel pfkelch13 Gene Polymorphism Associates With Artemisinin Resistance in Eastern India

Clinical Infectious Diseases ◽

10.1093/cid/ciy1038 ◽

2018 ◽

Vol 69 (7) ◽

pp. 1144-1152 ◽

Cited By ~ 15

Author(s):

Sabyasachi Das ◽

Subhankar Manna ◽

Bhaskar Saha ◽

Amiya Kumar Hati ◽

Somenath Roy

Keyword(s):

Combination Therapy ◽

Treatment Failure ◽

Ex Vivo ◽

Artemisinin Combination Therapy ◽

Gene Mutations ◽

Artemisinin Resistance ◽

Parasite Clearance ◽

World Health ◽

Ring Stage ◽

Self Medication

Abstract Background Artesunate-sulfadoxine-pyrimethamine (ASSP) is the frontline artemisinin combination therapy (ACT) in India. Random, irrational, subtherapeutic artemisinin doses and self-medication with ACT along with predominance of sulfadoxine-pyrimethamine resistance parasite invoked a strong possibility of emerging artemisinin-resistant malaria parasites. Methods This study involved 226 patients with uncomplicated Plasmodium falciparum infection who had successfully completed the 42 days follow-up after ASSP combination therapy from April 2014 to January 2016. We assessed the ASSP treatment efficacy by evaluating parasite clearance half-life, pfkelch13, and other (pfdhfr, pfdhps, pfmdr1, pfcrt) gene mutations and survival of parasites as detected by an ex vivo ring-stage survival assay (RSA). Findings Slow-clearing infections with longer parasite clearance half-lives (>5 hours) were observed in 12% isolates. Cure rate after ASSP treatment was declining to about 84.1%. ASSP failure was recorded in 15.9% (early treatment failure, 7.9%; late treatment failure, 7.9%) of isolates. In sum, 24 patients (10.6%) had parasite clearance half-lives greater than 5 hours with pfkelch13 polymorphism after 441 codon; in 15 of those patients (6.6%), parasites had not cleared by 72 hours after initiation of therapy. Median ex vivo ring-stage survival rate of these isolates was very high (12.2%; 95% confidence interval [CI], 10.9–13.8) from that of cured patients (0.9%; 95% CI, 0.09–1.07). Of these 15 patients, 13 patients had pfkelch13 G625R polymorphism, whereas 2 patients contained R539T polymorphism. As per the World Health Organization guideline, these 15 isolates were true artemisinin-resistant isolates. Interpretation Identification of artemisinin-resistant isolates in India together with new mutations and increasing combination therapy failures blow alarms for urgent malaria control.

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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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Atovaquone-Proguanil in Combination With Artesunate to Treat Multidrug-Resistant P. falciparum Malaria in Cambodia: An Open-Label Randomized Trial

Open Forum Infectious Diseases ◽

10.1093/ofid/ofz314 ◽

2019 ◽

Vol 6 (9) ◽

Cited By ~ 3

Author(s):

Mariusz Wojnarski ◽

Chanthap Lon ◽

Pattaraporn Vanachayangkul ◽

Panita Gosi ◽

Somethy Sok ◽

...

Keyword(s):

Low Dose ◽

Ex Vivo ◽

Artemisinin Combination Therapy ◽

Resistance Mutation ◽

Artemisinin Resistance ◽

Multidrug Resistant ◽

Fixed Dose ◽

Resistance Mutations ◽

Open Label

Abstract Background Recent artemisinin-combination therapy failures in Cambodia prompted a search for alternatives. Atovaquone-proguanil (AP), a safe, effective treatment for multidrug-resistant Plasmodium falciparum (P.f.), previously demonstrated additive effects in combination with artesunate (AS). Methods Patients with P.f. or mixed-species infection (n = 205) in Anlong Veng (AV; n = 157) and Kratie (KT; n = 48), Cambodia, were randomized open-label 1:1 to a fixed-dose 3-day AP regimen +/-3 days of co-administered artesunate (ASAP). Single low-dose primaquine (PQ, 15 mg) was given on day 1 to prevent gametocyte-mediated transmission. Results Polymerase chain reaction–adjusted adequate clinical and parasitological response at 42 days was 90% for AP (95% confidence interval [CI], 82%–95%) and 92% for ASAP (95% CI, 83%–96%; P = .73). The median parasite clearance time was 72 hours for ASAP in AV vs 56 hours in KT (P < .001) and was no different than AP alone. At 1 week postprimaquine, 7% of the ASAP group carried microscopic gametocytes vs 29% for AP alone (P = .0001). Nearly all P.f. isolates had C580Y K13 propeller artemisinin resistance mutations (AV 99%; KT 88%). Only 1 of 14 treatment failures carried the cytochrome bc1 (Pfcytb) atovaquone resistance mutation, which was not present at baseline. P.f. isolates remained atovaquone sensitive in vitro but cycloguanil resistant, with a triple P.f. dihydrofolate reductase mutation. Conclusions Atovaquone-proguanil remained marginally effective in Cambodia (≥90%) with minimal Pfcytb mutations observed. Treatment failures in the presence of ex vivo atovaquone sensitivity and adequate plasma levels may be attributable to cycloguanil and/or artemisinin resistance. Artesunate co-administration provided little additional blood-stage efficacy but reduced post-treatment gametocyte carriage in combination with AP beyond single low-dose primaquine.

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Efficacy of dihydroartemisinin/piperaquine and artesunate monotherapy for the treatment of uncomplicated Plasmodium falciparum malaria in Central Vietnam

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkaa172 ◽

2020 ◽

Author(s):

Eduard Rovira-Vallbona ◽

Nguyen Van Hong ◽

Johanna H Kattenberg ◽

Ro Mah Huan ◽

Nguyen Thi Thu Hien ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Falciparum Malaria ◽

Ex Vivo ◽

Survival Rates ◽

Artemisinin Resistance ◽

Plasmodium Falciparum Malaria ◽

Parasite Clearance ◽

Resistance Mutations ◽

Malaria Burden ◽

Central Vietnam

Abstract Background Artemisinin-based combination therapies (ACTs) have significantly contributed to reduce Plasmodium falciparum malaria burden in Vietnam, but their efficacy is challenged by treatment failure of dihydroartemisinin/piperaquine ACT in Southern provinces. Objectives To assess the efficacy of dihydroartemisinin/piperaquine for uncomplicated P. falciparum malaria in Gia Lai, Central Vietnam, and determine parasite resistance to artemisinin (ClinicalTrials.gov identifier NCT02604966). Methods Sixty patients received either dihydroartemisinin/piperaquine (4 mg/kg/day, 3 days; n = 33) or artesunate monotherapy (4 mg/kg/day, 3 days; n = 27) followed by dihydroartemisinin/piperaquine (AS + DHA/PPQ). Clinical phenotypes were determined during a 42 day follow-up and analysed together with ex vivo susceptibility to antimalarials and molecular markers of drug resistance. Results Day 3 positivity rate was significantly higher in the AS + DHA/PPQ arm compared with dihydroartemisinin/piperaquine (70.4% versus 39.4%, P = 0.016). Parasite clearance time was 95.2 h (AS + DHA/PPQ) versus 71.9 h (dihydroartemisinin/piperaquine, P = 0.063) and parasite clearance half-life was 7.4 h (AS + DHA/PPQ) versus 7.0 h (dihydroartemisinin/piperaquine, P = 0.140). Adequate clinical and parasitological response at Day 42 was 100% in both arms. By RT–qPCR, 36% (19/53) patients remained positive until Day 7. No recurrences were detected. kelch13 artemisinin resistance mutations were found in 87% (39/45) of isolates and 50% (20/40) were KEL1/C580Y. The piperaquine resistance marker plasmepsin-2 was duplicated in 10.4% (5/48). Isolates from Day 3-positive patients (n = 18) had higher ex vivo survival rates to artemisinin compounds (P < 0.048) and prevalence of kelch13 mutations (P = 0.005) than Day 3-negative patients (n = 5). The WHO definition of artemisinin resistance was fulfilled in 60% (24/40) of cases. Conclusions Although dihydroartemisinin/piperaquine remained effective to treat P. falciparum, the high Day 3 positivity rate and prevalence of KEL1 strains calls for continuous monitoring of dihydroartemisinin/piperaquine efficacy in Central Vietnam.

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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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Sustained Ex Vivo Susceptibility of Plasmodium falciparum to Artemisinin Derivatives but Increasing Tolerance to Artemisinin Combination Therapy Partner Quinolines in The Gambia

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00759-17 ◽

2017 ◽

Vol 61 (12) ◽

Cited By ~ 9

Author(s):

Alfred Amambua-Ngwa ◽

Joseph Okebe ◽

Haddijatou Mbye ◽

Sukai Ceesay ◽

Fatima El-Fatouri ◽

...

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Combination Therapy ◽

Genomic Sequence ◽

Sequence Data ◽

Ex Vivo ◽

Artemisinin Combination Therapy ◽

Artemisinin Resistance ◽

The Gambia ◽

Microsatellite Variation

ABSTRACT Antimalarial interventions have yielded a significant decline in malaria prevalence in The Gambia, where artemether-lumefantrine (AL) has been used as a first-line antimalarial for a decade. Clinical Plasmodium falciparum isolates collected from 2012 to 2015 were analyzed ex vivo for antimalarial susceptibility and genotyped for drug resistance markers (pfcrt K76T, pfmdr1 codons 86, 184, and 1246, and pfk13) and microsatellite variation. Additionally, allele frequencies of single nucleotide polymorphisms (SNPs) from other drug resistance-associated genes were compared from genomic sequence data sets from 2008 (n = 79) and 2014 (n = 168). No artemisinin resistance-associated pfk13 mutation was found, and only 4% of the isolates tested in 2015 showed significant growth after exposure to dihydroartemisinin. Conversely, the 50% inhibitory concentrations (IC50s) of amodiaquine and lumefantrine increased within this period. pfcrt 76T and pfmdr1 184F mutants remained at a prevalence above 80%. pfcrt 76T was positively associated with higher IC50s to chloroquine. pfmdr1 NYD increased in frequency between 2012 and 2015 due to lumefantrine selection. The TNYD (pfcrt 76T and pfmdr1 NYD wild-type haplotype) also increased in frequency following AL implementation in 2008. These results suggest selection for pfcrt and pfmdr1 genotypes that enable tolerance to lumefantrine. Increased tolerance to lumefantrine calls for sustained chemotherapeutic monitoring in The Gambia to minimize complete artemisinin combination therapy (ACT) failure in the future.

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Artemisinin Therapy for Malaria in Hemoglobinopathies: A Systematic Review

Clinical Infectious Diseases ◽

10.1093/cid/cix785 ◽

2018 ◽

Vol 66 (5) ◽

pp. 799-804 ◽

Cited By ~ 3

Author(s):

Sri Riyati Sugiarto ◽

Brioni R Moore ◽

Julie Makani ◽

Timothy M E Davis

Keyword(s):

Clinical Studies ◽

Antimalarial Activity ◽

Artemisinin Combination Therapy ◽

Plasma Concentrations ◽

Parasite Clearance ◽

Convincing Evidence ◽

Hemoglobin E ◽

Artemisinin Derivatives

Abstract Artemisinin derivatives are widely used antimalarial drugs. There is some evidence from in vitro, animal and clinical studies that hemoglobinopathies may alter their disposition and antimalarial activity. This review assesses relevant data in α-thalassemia, sickle cell disease (SCD), β-thalassemia and hemoglobin E. There is no convincing evidence that the disposition of artemisinin drugs is affected by hemoglobinopathies. Although in vitro studies indicate that Plasmodium falciparum cultured in thalassemic erythrocytes is relatively resistant to the artemisinin derivatives, mean 50% inhibitory concentrations (IC50s) are much lower than in vivo plasma concentrations after recommended treatment doses. Since IC50s are not increased in P. falciparum cultures using SCD erythrocytes, delayed post-treatment parasite clearance in SCD may reflect hyposplenism. As there have been no clinical studies suggesting that hemoglobinopathies significantly attenuate the efficacy of artemisinin combination therapy (ACT) in uncomplicated malaria, recommended artemisinin doses as part of ACT remain appropriate in this patient group.

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Anthrahydroquinone-2-6-disulfonate is a novel, powerful antidote for paraquat poisoning

Scientific Reports ◽

10.1038/s41598-021-99591-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Jin Qian ◽

Chun-Yuan Wu ◽

Dong-Ming Wu ◽

Li-Hua Li ◽

Qi Li ◽

...

Keyword(s):

Ex Vivo ◽

A549 Cells ◽

Organ Damage ◽

Self Administration ◽

Nextgen Sequencing ◽

Animal Survival ◽

Detoxification Mechanism ◽

Stress Damage ◽

Fast Acting

AbstractParaquat (PQ) is a widely used fast-acting pyridine herbicide. Accidental ingestion or self-administration via various routes can cause severe organ damage. Currently, no effective antidote is available commercially, and the mortality rate of poisoned patients is exceptionally high. Here, the efficacy of anthrahydroquinone-2-6-disulfonate (AH2QDS) was observed in treating PQ poisoning by constructing in vivo and ex vivo models. We then explored the detoxification mechanism of AH2QDS. We demonstrated that, in a rat model, the PQ concentration in the PQ + AH2QDS group significantly decreased compared to the PQ only group. Additionally, AH2QDS protected the mitochondria of rats and A549 cells and decreased oxidative stress damage, thus improving animal survival and cell viability. Finally, the differentially expressed genes were analysed in the PQ + AH2QDS group and the PQ group by NextGen sequencing, and we verified that Nrf2’s expression in the PQ + AH2QDS group was significantly higher than that in the PQ group. Our work identified that AH2QDS can detoxify PQ by reducing PQ uptake and protecting mitochondria while enhancing the body's antioxidant activity.

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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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