The Artemisinin Resistance in Southeast Asia: An Imminent Global Threat to Malaria Elimination

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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Surveillance of genetic markers associated with Plasmodium falciparum resistance to artemisinin-based combination therapy in Pakistan, 2018-2019

10.21203/rs.3.rs-16248/v1 ◽

2020 ◽

Author(s):

Abdul Qader Khan ◽

Leyre Pernaute-Lau ◽

Aamer Ali Khattak ◽

Sanna Luijcx ◽

Berit Aydin-Schmidt ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Molecular Markers ◽

Southeast Asia ◽

Treatment Guidelines ◽

Treatment Guideline ◽

Artemisinin Resistance ◽

Chloroquine Resistance ◽

Close Monitoring ◽

Malaria Surveillance ◽

Current Prevalence

Abstract Background: The spread of artemisinin resistance in the Greater Mekong Subregion of Southeast Asia poses a significant threat for current anti-malarial treatment guidelines globally. The aim of this study was to assess the current prevalence of molecular markers of drug resistance in Plasmodium falciparum in the four provinces with the highest malaria burden in Pakistan, after introducing artemether-lumefantrine as first line treatment in 2017.Methods: Samples were collected during routine malaria surveillance in Punjab, Sindh, Baluchistan, and Khyber Pakhtunkhwa provinces of Pakistan between January 2018 and February 2019. P. falciparum infections were confirmed by rapid diagnostic test or microscopy. P. falciparum positive isolates (n = 179) were screened by Sanger sequencing for single nucleotide polymorphisms (SNPs) in the P. falciparum kelch 13 (pfk13) propeller domain and in P. falciparum coronin (pfcoronin). SNPs in P. falciparum multidrug resistance 1 (pfmdr1) N86Y, Y184F, D1246Y and P. falciparum chloroquine resistance transporter (pfcrt) K76T were genotyped by PCR-restriction fragment length polymorphism. Results: No artemisinin resistance associated SNPs were identified in the pfk13 propeller domain or in pfcoronin. The pfmdr1 N86, 184F, D1246 and pfcrt K76 alleles associated with reduced lumefantrine sensitivity were present in 83.8% (150/179), 16.9% (29/172), 100.0% (173/173), and 8.4% (15/179) of all infections, respectively. The chloroquine resistance associated pfcrt 76T allele was present in 98.3% (176/179) of infections.Conclusion: This study provides an update on the current prevalence of molecular markers associated with reduced sensitivity to artemether and/or lumefantrine in P. falciparum, including a first baseline assessment of polymorphisms in pfcoronin. No mutations associated with artemisinin resistance were observed in pfk13 or pfcoronin. However, the prevalence of the pfmdr1 N86 and D1246 alleles, that have been associated with decreased susceptibility to lumefantrine, remain high. Although clinical and molecular data suggest that the current malaria treatment guideline for P. falciparum are presently effective in Pakistan, close monitoring for artemisinin and lumefantrine resistance will be critical to ensure early detection and enhanced containment of emerging ACT resistance spreading across from Southeast Asia.

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

10.21203/rs.2.18721/v1 ◽

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.

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Preparing for the Next Global Threat: A Call for Targeted, Immediate Decisive Action in Southeast Asia to Prevent the Next Pandemic in Africa

Towards Malaria Elimination - A Leap Forward ◽

10.5772/intechopen.78261 ◽

2018 ◽

Author(s):

Colin Ohrt ◽

Thang Duc Ngo ◽

Thieu Quang Nguyen

Keyword(s):

Southeast Asia ◽

Decisive Action ◽

Global Threat

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Experimentally Engineered Mutations in a Ubiquitin Hydrolase, UBP-1, Modulate In Vivo Susceptibility to Artemisinin and Chloroquine in Plasmodium berghei

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02484-19 ◽

2020 ◽

Vol 64 (7) ◽

Cited By ~ 3

Author(s):

Nelson V. Simwela ◽

Katie R. Hughes ◽

A. Brett Roberts ◽

Michael T. Rennie ◽

Michael P. Barrett ◽

...

Keyword(s):

Southeast Asia ◽

Plasmodium Berghei ◽

Molecular Mechanisms ◽

Natural Infection ◽

Artemisinin Resistance ◽

Growth Defect ◽

Genetic Determinants ◽

Content Type ◽

Mutant Lines

ABSTRACT As resistance to artemisinins (current frontline drugs in malaria treatment) emerges in Southeast Asia, there is an urgent need to identify the genetic determinants and understand the molecular mechanisms underpinning such resistance. Such insights could lead to prospective interventions to contain resistance and prevent the eventual spread to other regions where malaria is endemic. Reduced susceptibility to artemisinin in Southeast Asia has been primarily linked to mutations in the Plasmodium falciparum Kelch-13 gene, which is currently widely recognized as a molecular marker of artemisinin resistance. However, two mutations in a ubiquitin hydrolase, UBP-1, have been previously associated with reduced artemisinin susceptibility in a rodent model of malaria, and some cases of UBP-1 mutation variants associated with artemisinin treatment failure have been reported in Africa and SEA. In this study, we employed CRISPR-Cas9 genome editing and preemptive drug pressures to test these artemisinin susceptibility-associated mutations in UBP-1 in Plasmodium berghei sensitive lines in vivo. Using these approaches, we show that the V2721F UBP-1 mutation results in reduced artemisinin susceptibility, while the V2752F mutation results in resistance to chloroquine (CQ) and moderately impacts tolerance to artemisinins. Genetic reversal of the V2752F mutation restored chloroquine sensitivity in these mutant lines, whereas simultaneous introduction of both mutations could not be achieved and appears to be lethal. Interestingly, these mutations carry a detrimental growth defect, which would possibly explain their lack of expansion in natural infection settings. Our work provides independent experimental evidence on the role of UBP-1 in modulating parasite responses to artemisinin and chloroquine under in vivo conditions.

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The promise, problems and pitfalls of mass drug administration for malaria elimination: a qualitative study with scientists and policymakers

International Health ◽

10.1093/inthealth/ihy079 ◽

2018 ◽

Vol 11 (3) ◽

pp. 166-176 ◽

Cited By ~ 9

Author(s):

Nils Kaehler ◽

Bipin Adhikari ◽

Phaik Yeong Cheah ◽

Nicholas P J Day ◽

Daniel H Paris ◽

...

Keyword(s):

Drug Administration ◽

Mass Drug Administration ◽

Malaria Elimination ◽

Artemisinin Resistance ◽

Malaria Prevention ◽

Evidence Base ◽

World Health ◽

Pilot Studies ◽

Target Populations ◽

Health Organization

Abstract Background The emergence of artemisinin resistance in the Greater Mekong Subregion (GMS) has prompted urgent containment measures. One possible approach is mass drug administration (MDA). This article explores attitudes towards and perceptions of MDA for malaria elimination among policymakers and leading malariologists. Methods Thirty-two semistructured interviews (SSI) were conducted with policymakers (n=17) and principal investigators (n=15) selected based on their involvement in malaria prevention, control and elimination in the GMS. Interviews were audio recorded and transcribed for qualitative content (thematic) analysis using NVivo (QSR International, Doncaster, Victoria, Australia). Results Researchers and policymakers described reluctance and consequently delays to pilot MDA for malaria elimination. Most policymakers and some researchers reported concerns around the evidence base, citing a lack of data on its effectiveness and appropriate target populations. There were also worries about promoting resistance. Other issues included a previous lack of support from the World Health Organization, past MDAs, the remoteness of target populations and challenges explaining the rationale for MDA. Conclusions The complex rationale for MDA for malaria elimination, mistaking pilot studies for implementation, past experiences with MDA, difficulties in selecting appropriate sites and the WHO’s lack of clear backing undermined the support for MDA for malaria elimination.

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Independent Emergence of Artemisinin Resistance Mutations Among Plasmodium falciparum in Southeast Asia

The Journal of Infectious Diseases ◽

10.1093/infdis/jiu491 ◽

2014 ◽

Vol 211 (5) ◽

pp. 670-679 ◽

Cited By ~ 257

Author(s):

Shannon Takala-Harrison ◽

Christopher G. Jacob ◽

Cesar Arze ◽

Michael P. Cummings ◽

Joana C. Silva ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Southeast Asia ◽

Artemisinin Resistance ◽

Resistance Mutations

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Limited Polymorphism of the Kelch Propeller Domain in Plasmodium malariae and P. ovale Isolates from Thailand

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00138-16 ◽

2016 ◽

Vol 60 (7) ◽

pp. 4055-4062 ◽

Cited By ~ 3

Author(s):

Supatchara Nakeesathit ◽

Naowarat Saralamba ◽

Sasithon Pukrittayakamee ◽

Arjen Dondorp ◽

Francois Nosten ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Southeast Asia ◽

Severe Malaria ◽

Malaria Control ◽

Artemisinin Resistance ◽

Plasmodium Malariae ◽

The Other ◽

Content Type ◽

Functional Consequences ◽

First Time

ABSTRACTArtemisinin resistance inPlasmodium falciparum, the agent of severe malaria, is currently a major obstacle to malaria control in Southeast Asia. A gene named “kelch13” has been associated with artemisinin resistance inP. falciparum. The orthologue of thekelchgene inP. vivaxwas identified and a small number of mutations were found in previous studies. Thekelchorthologues in the other two human malaria parasites,P. malariaeandP. ovale, have not yet been studied. Therefore, in this study, the orthologouskelchgenes ofP. malariae,P. ovale wallikeri, andP. ovale curtisiwere isolated and analyzed for the first time. The homologies of thekelchgenes ofP. malariaeandP. ovalewere 84.8% and 82.7%, respectively, compared to the gene inP. falciparum.kelchpolymorphisms were studied in 13P. malariaeand 5P. ovaleisolates from Thailand. There were 2 nonsynonymous mutations found in these samples. One mutation was P533L, which was found in 1 of 13P. malariaeisolates, and the other was K137R, found in 1 isolate ofP. ovale wallikeri(n= 4). This result needs to be considered in the context of widespread artemisinin used within the region; their functional consequences for artemisinin sensitivity inP. malariaeandP. ovalewill need to be elucidated.

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