Search

The emergence of Plasmodium falciparum parasites resistant to artemisinins compromises the efficacy of Artemisinin Combination Therapies (ACTs), the global first-line malaria treatment. Artemisinin resistance is a complex genetic trait in which nonsynonymous SNPs in PfK13 cooperate with other genetic variations. Here, we present population genomic/transcriptomic analyses of P. falciparum collected from patients with uncomplicated malaria in Cambodia and Vietnam between 2018 and 2020. Besides the PfK13 SNPs, several polymorphisms, including nonsynonymous SNPs (N1131I and N821K) in PfRad5 and an intronic SNP in PfWD11 (WD40 repeat-containing protein on chromosome 11), appear to be associated with artemisinin resistance, possibly as new markers. There is also a defined set of genes whose steady-state levels of mRNA and/or splice variants or antisense transcripts correlate with artemisinin resistance at the base level. In vivo transcriptional responses to artemisinins indicate the resistant parasite's capacity to decelerate its intraerythrocytic developmental cycle (IDC), which can contribute to the resistant phenotype. During this response, PfRAD5 and PfWD11 upregulate their respective alternatively/aberrantly spliced isoforms, suggesting their contribution to the protective response to artemisinins. PfRAD5 and PfWD11 appear under selective pressure in the Greater Mekong Sub-region over the last decade, suggesting their role in the genetic background of the artemisinin resistance., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Piperaquine (PPQ) is widely used in combination with dihydroartemisinin as a first-line treatment against malaria. Multiple genetic drivers of PPQ resistance have been reported, including mutations in the Plasmodium falciparum chloroquine resistance transporter ( pfcrt ) and increased copies of plasmepsin II/III ( pm2/3 ). We generated a cross between a Cambodia-derived multidrug-resistant KEL1/PLA1 lineage isolate (KH004) and a drug-susceptible Malawian parasite (Mal31). Mal31 harbors a wild-type (3D7-like) pfcrt allele and a single copy of pm2/3 , while KH004 has a chloroquine-resistant (Dd2-like) pfcrt allele with an additional G367C substitution and multiple copies of pm2/3 . We recovered 104 unique recombinant parasites and examined a targeted set of progeny representing all possible combinations of variants at pfcrt and pm2/3 . We performed a detailed analysis of competitive fitness and a range of PPQ susceptibility phenotypes with these progenies, including PPQ survival assay, area under the dose response curve, and a limited point IC 50 . We find that inheritance of the KH004 pfcrt allele is required for reduced PPQ sensitivity, whereas copy number variation in pm2/3 further decreases susceptibility but does not confer resistance in the absence of additional mutations in pfcrt . A deep investigation of genotype-phenotype relationships demonstrates that progeny clones from experimental crosses can be used to understand the relative contributions of pfcrt , pm2/3 , and parasite genetic background to a range of PPQ-related traits. Additionally, we find that the resistance phenotype associated with parasites inheriting the G367C substitution in pfcrt is consistent with previously validated PPQ resistance mutations in this transporter.IMPORTANCEResistance to piperaquine, used in combination with dihydroartemisinin, has emerged in Cambodia and threatens to spread to other malaria-endemic regions. Understanding the causal mutations of drug resistance and their impact on parasite fitness is critical for surveillance and intervention and can also reveal new avenues to limiting the evolution and spread of drug resistance. An experimental genetic cross is a powerful tool for pinpointing the genetic determinants of key drug resistance and fitness phenotypes and has the distinct advantage of quantifying the effects of naturally evolved genetic variation. Our study was strengthened since the full range of copies of KH004 pm2/3 was inherited among the progeny clones, allowing us to directly test the role of the pm2/3 copy number on resistance-related phenotypes in the context of a unique pfcrt allele. Our multigene model suggests an important role for both loci in the evolution of this multidrug-resistant parasite lineage., Competing Interests: The authors declare no conflict of interest.

Background: Newly emerged mutations within the Plasmodium falciparum chloroquine resistance transporter (PfCRT) can confer piperaquine resistance in the absence of amplified plasmepsin II (pfpm2). In this study, we estimated the prevalence of co-circulating piperaquine resistance mutations in P. falciparum isolates collected in northern Cambodia from 2009 to 2017., Methods: The sequence of pfcrt was determined for 410 P. falciparum isolates using PacBio amplicon sequencing or whole genome sequencing. Quantitative polymerase chain reaction was used to estimate pfpm2 and pfmdr1 copy number., Results: Newly emerged PfCRT mutations increased in prevalence after the change to dihydroartemisinin-piperaquine in 2010, with >98% of parasites harboring these mutations by 2017. After 2014, the prevalence of PfCRT F145I declined, being outcompeted by parasites with less resistant, but more fit PfCRT alleles. After the change to artesunate-mefloquine, the prevalence of parasites with amplified pfpm2 decreased, with nearly half of piperaquine-resistant PfCRT mutants having single-copy pfpm2., Conclusions: The large proportion of PfCRT mutants that lack pfpm2 amplification emphasizes the importance of including PfCRT mutations as part of molecular surveillance for piperaquine resistance in this region. Likewise, it is critical to monitor for amplified pfmdr1 in these PfCRT mutants, as increased mefloquine pressure could lead to mutants resistant to both drugs., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Background: National Malaria Control Programmes (NMCPs) currently make limited use of parasite genetic data. We have developed GenRe-Mekong, a platform for genetic surveillance of malaria in the Greater Mekong Subregion (GMS) that enables NMCPs to implement large-scale surveillance projects by integrating simple sample collection procedures in routine public health procedures., Methods: Samples from symptomatic patients are processed by SpotMalaria, a high-throughput system that produces a comprehensive set of genotypes comprising several drug resistance markers, species markers and a genomic barcode. GenRe-Mekong delivers Genetic Report Cards, a compendium of genotypes and phenotype predictions used to map prevalence of resistance to multiple drugs., Results: GenRe-Mekong has worked with NMCPs and research projects in eight countries, processing 9623 samples from clinical cases. Monitoring resistance markers has been valuable for tracking the rapid spread of parasites resistant to the dihydroartemisinin-piperaquine combination therapy. In Vietnam and Laos, GenRe-Mekong data have provided novel knowledge about the spread of these resistant strains into previously unaffected provinces, informing decision-making by NMCPs., Conclusions: GenRe-Mekong provides detailed knowledge about drug resistance at a local level, and facilitates data sharing at a regional level, enabling cross-border resistance monitoring and providing the public health community with valuable insights. The project provides a rich open data resource to benefit the entire malaria community., Funding: The GenRe-Mekong project is funded by the Bill and Melinda Gates Foundation (OPP11188166, OPP1204268). Genotyping and sequencing were funded by the Wellcome Trust (098051, 206194, 203141, 090770, 204911, 106698/B/14/Z) and Medical Research Council (G0600718). A proportion of samples were collected with the support of the UK Department for International Development (201900, M006212), and Intramural Research Program of the National Institute of Allergy and Infectious Diseases., Competing Interests: CJ, NT, MM, RM, HQ, BH, VV, TN, HR, Rv, Lv, RF, FN, MH, NP, SG, PR, KC, PN, EA, SP, RM, RL, CH, LD, KN, TN, TH, AS, DU, CB, CF, MO, TP, RT, CA, AM, GD, JL, DP, NC, DL, SS, JC, PJ, BH, SP, AP, FS, KL, MT, TH, PS, SS, PB, AT, SB, NV, AA, AU, AF, CK, ED, MK, MA, KL, SR, AJ, KR, CH, MD, RV, NK, PW, JA, RP, CA, TC, CM, TN, JS, BJ, JK, KJ, DM, XC, JS, RA, VS, SG, KR, ND, AD, DK, OM No competing interests declared, HN, NZ, SC is an employee of Vysnova Partners Inc

Background: Plasmodium vivax resistance to chloroquine (CQ) has been reported worldwide, although the World Health Organization clinical drug efficacy studies protocol does not permit classification of patient outcomes., Methods: We enrolled 40 patients with P. vivax malaria in northeastern Cambodia, where >17% treatment failures were previously reported. Patients were treated with CQ (30 mg/kg) and followed for 2 months, with frequent clinical examination and capillary blood sample collection for microscopy, molecular parasite detection and genotyping, and drug concentration measurements. Reinfections were prevented by relocating patients to a transmission-free area., Results: P. vivax parasites were eliminated in all patients by day 3. Genomic analyses revealed that all clones in polyclonal infections were cleared at the same rate, indicating their equal susceptibility to CQ. CQ blood concentrations were below the therapeutic level in all recurrent infections (24 of 40 patients), which were efficiently cleared by a second course of CQ treatment. Genotyping (128 SNPs barcode) and sequences of entire parasite genome (Whole-Genome Sequencing, Illumina) indicated that two thirds (6 of 8) of the recurrent parasites resulted from heterologous relapses whose 50% are from by sibling/recombinant clones., Conclusions: No evidence of CQ resistance was observed. Our data suggest that P. vivax antimalarial drug resistance is likely overestimated and that the current guidelines for clinical drug studies of P. vivax malaria need to be revised.

Background: In a drug-resistant, malaria elimination setting like Western Cambodia, field research is essential for the development of novel anti-malarial regimens and the public health solutions necessary to monitor the spread of resistance and eliminate infection. Such field studies often face a variety of similar implementation challenges, but these are rarely captured in a systematic way or used to optimize future study designs that might overcome similar challenges. Field-based research staff often have extensive experience and can provide valuable insight regarding these issues, but their perspectives and experiences are rarely documented and seldom integrated into future research protocols. This mixed-methods analysis sought to gain an understanding of the daily challenges encountered by research field staff in the artemisinin-resistant, malaria elimination setting of Western Cambodia. In doing so, this study seeks to understand how the experiences and opinions of field staff can be captured, and used to inform future study designs., Methods: Twenty-two reports from six field-based malaria studies conducted in Western Cambodia were reviewed using content analysis to identify challenges to conducting the research. Informal Interviews, Focus Group Discussions and In-depth Interviews were also conducted among field research staff. Thematic analysis of the data was undertaken using Nvivo 9 ® software. Triangulation and critical case analysis was also used., Results: There was a lack of formalized avenues through which field workers could report challenges experienced when conducting the malaria studies. Field research staff faced significant logistical barriers to participant recruitment and data collection, including a lack of available transportation to cover long distances, and the fact that mobile and migrant populations (MMPs) are usually excluded from studies because of challenges in follow-up. Cultural barriers to communication also hindered participant recruitment and created unexpected delays. Field staff often paid a physical, emotional and financial cost, going beyond their duty in order to keep the study running., Conclusions: Formal monthly reports filled out by field study staff could be a key tool for capturing field study staff experiences effectively, but require specific report fields to encourage staff to outline their challenges and to propose potential solutions. Forging strong bonds with communities and their leaders may improve communication, and decrease barriers to participant recruitment. Study designs that make it feasible for MMPs to participate should be pursued; in addition to increasing the potential participant pool, this will ensure that the most malaria-endemic demographic is taken into account in research studies. Overlaps between clinical care and research create ethical dilemmas for study staff, a fact that warrants careful consideration. Lessons learned from study field staff should be used to create a set of locally-relevant recommendations to inform future study designs.

Background: Western Cambodia is recognized as the epicentre of emergence of Plasmodium falciparum multi-drug resistance. The emergence of artemisinin resistance has been observed in this area since 2008-2009 and molecular signatures associated to artemisinin resistance have been characterized in k13 gene. At present, one of the major threats faced, is the possible spread of Asian artemisinin resistant parasites over the world threatening millions of people and jeopardizing malaria elimination programme efforts. To anticipate the diffusion of artemisinin resistance, the identification of the P. falciparum population structure and the gene flow among the parasite population in Cambodia are essential., Methods: To this end, a mid-throughput PCR-LDR-FMA approach based on LUMINEX technology was developed to screen for genetic barcode in 533 blood samples collected in 2010-2011 from 16 health centres in malaria endemics areas in Cambodia., Results: Based on successful typing of 282 samples, subpopulations were characterized along the borders of the country. Each 11-loci barcode provides evidence supporting allele distribution gradient related to subpopulations and gene flow. The 11-loci barcode successfully identifies recently emerging parasite subpopulations in western Cambodia that are associated with the C580Y dominant allele for artemisinin resistance in k13 gene. A subpopulation was identified in northern Cambodia that was associated to artemisinin (R539T resistant allele of k13 gene) and mefloquine resistance., Conclusions: The gene flow between these subpopulations might have driven the spread of artemisinin resistance over Cambodia.

BackgroundNational Malaria Control Programmes (NMCPs) currently make limited use of parasite genetic data. We have developed GenRe-Mekong, a platform for genetic surveillance of malaria in the Greater Mekong Subregion (GMS) that enables NMCPs to implement large-scale surveillance projects by integrating simple sample collection procedures in routine public health procedures.MethodsSamples from symptomatic patients are processed by SpotMalaria, a high-throughput system that produces a comprehensive set of genotypes comprising several drug resistance markers, species markers and a genomic barcode. GenRe-Mekong delivers Genetic Report Cards, a compendium of genotypes and phenotype predictions used to map prevalence of resistance to multiple drugs.ResultsGenRe-Mekong has worked with NMCPs and research projects in eight countries, processing 9623 samples from clinical cases. Monitoring resistance markers has been valuable for tracking the rapid spread of parasites resistant to the dihydroartemisinin-piperaquine combination therapy. In Vietnam and Laos, GenRe-Mekong data have provided novel knowledge about the spread of these resistant strains into previously unaffected provinces, informing decision-making by NMCPs.ConclusionsGenRe-Mekong provides detailed knowledge about drug resistance at a local level, and facilitates data sharing at a regional level, enabling cross-border resistance monitoring and providing the public health community with valuable insights. The project provides a rich open data resource to benefit the entire malaria community.FundingThe GenRe-Mekong project is funded by the Bill and Melinda Gates Foundation (OPP11188166, OPP1204268). Genotyping and sequencing were funded by the Wellcome Trust (098051, 206194, 203141, 090770, 204911, 106698/B/14/Z) and Medical Research Council (G0600718). A proportion of samples were collected with the support of the UK Department for International Development (201900, M006212), and Intramural Research Program of the National Institute of Allergy and Infectious Diseases.

Background: During the process of malaria elimination in the Greater Mekong Sub-region, mobile and migrant populations (MMPs) have been identified as the most at-risk demographic. An important sub-group of MMPs are seasonal workers, and this paper presents an evaluation of the reach and effectiveness of interventions tailored towards this group and was carried out as part of the Containment Project from 2009-11. Methods: A mixed-methods study was conducted in Pailin Province in Western Cambodia. Three-hundred-and-four seasonal workers were surveyed using a structured questionnaire. Qualitative data were gathered through a total of eight focus group discussions and 14 in-depth interviews. Data triangulation of the qualitative and quantitative data was used during analysis. Results: High mobility and low access of the target population to the interventions, as well as lack of social and anthropological research that led to implementation oversights, resulted in under-exposure of seasonal workers to interventions. Consequently, their reach and impact were severely limited. Some services, particularly Mobile Malaria Workers, had the ability to significantly impact key factors, such as risky behaviours among those they did reach. Others, like Listening and Viewing Clubs and mass media campaigns, showed little impact. Conclusions: There is potential in two of the interventions assessed, but high mobility and inadequate exposure of seasonal workers to these interventions must be considered in the development and planning of future interventions to avoid investing in low-impact activities and ensure that all interventions perform according to their maximum potential. This will be critical in order for Cambodia to achieve its aim of malaria elimination. The lessons learned from this study can be extrapolated to other areas of health care in Cambodia and other countries in order to reduce the gap between healthcare provided to MMPs, especially seasonal workers, and to the general population. [ABSTRACT FROM AUTHOR]

Background: Countries of the greater Mekong subregion have made a transition from malaria control to an aim for falciparum and vivax malaria elimination. The elimination of falciparum malaria will have to be achieved against a background of increasing artemisinin and multi-drug resistance. This ambitious goal requires an operational research (OR) agenda that addresses the dynamic challenges encountered on the path to elimination, which will need to be flexible and developed in close relation with the cambodian national programme for parasitology, entomology and malaria control (CNM). In Cambodia, a number of meetings with stakeholders were convened by the CNM and emergency response to artemisinin resistance (ERAR) hub, producing an initial list of priority OR topics. The process and outcome of these meetings are described, which could serve as a template for other countries in the region. Methods: A landscaping exercise was conducted to gather all past, on-going and planned malaria focussed OR activities conducted by the cambodian research consortium in Cambodia and categorized according to research theme. The six themes included (1) malaria epidemiology, surveillance and response, (2) malaria case management, (3) malaria vector control, (4) malaria behavioural issues, (5) malaria clinical studies, and (6) other vector-borne diseases (dengue, neglected tropical diseases, soil-transmitted helminths). The different themes were discussed in small focus groups, which made an initial prioritization list which was then presented to a plenary group for further discussion. This produced a list of research questions ranked according to priority. Results: OR priorities produced by the thematic groups were discussed in the plenary meeting and given a priority score by group voting. A list of 17 OR questions were developed, finalized and listed, which included questions on surveillance, active case detection and treatment efficacy. Conclusion: This paper describes ERAR's work on supporting Cambodia's transition to malaria elimination by identifying national operational research priorities. ERAR has initiated and currently plays a critical role in the development of country specific research agendas for malaria elimination. The first example of this has been the described exercise in Cambodia, which could serve a template for setting OR priorities in the wider region. [ABSTRACT FROM AUTHOR]

Background: Artemisinin and partner-drug resistance in Plasmodium falciparum are major threats to malaria control and elimination. Triple artemisinin-based combination therapies (TACTs), which combine existing co-formulated ACTs with a second partner drug that is slowly eliminated, might provide effective treatment and delay emergence of antimalarial drug resistance.Methods: In this multicentre, open-label, randomised trial, we recruited patients with uncomplicated P falciparum malaria at 18 hospitals and health clinics in eight countries. Eligible patients were aged 2-65 years, with acute, uncomplicated P falciparum malaria alone or mixed with non-falciparum species, and a temperature of 37·5°C or higher, or a history of fever in the past 24 h. Patients were randomly assigned (1:1) to one of two treatments using block randomisation, depending on their location: in Thailand, Cambodia, Vietnam, and Myanmar patients were assigned to either dihydroartemisinin-piperaquine or dihydroartemisinin-piperaquine plus mefloquine; at three sites in Cambodia they were assigned to either artesunate-mefloquine or dihydroartemisinin-piperaquine plus mefloquine; and in Laos, Myanmar, Bangladesh, India, and the Democratic Republic of the Congo they were assigned to either artemether-lumefantrine or artemether-lumefantrine plus amodiaquine. All drugs were administered orally and doses varied by drug combination and site. Patients were followed-up weekly for 42 days. The primary endpoint was efficacy, defined by 42-day PCR-corrected adequate clinical and parasitological response. Primary analysis was by intention to treat. A detailed assessment of safety and tolerability of the study drugs was done in all patients randomly assigned to treatment. This study is registered at ClinicalTrials.gov, NCT02453308, and is complete.Findings: Between Aug 7, 2015, and Feb 8, 2018, 1100 patients were given either dihydroartemisinin-piperaquine (183 [17%]), dihydroartemisinin-piperaquine plus mefloquine (269 [24%]), artesunate-mefloquine (73 [7%]), artemether-lumefantrine (289 [26%]), or artemether-lumefantrine plus amodiaquine (286 [26%]). The median age was 23 years (IQR 13 to 34) and 854 (78%) of 1100 patients were male. In Cambodia, Thailand, and Vietnam the 42-day PCR-corrected efficacy after dihydroartemisinin-piperaquine plus mefloquine was 98% (149 of 152; 95% CI 94 to 100) and after dihydroartemisinin-piperaquine was 48% (67 of 141; 95% CI 39 to 56; risk difference 51%, 95% CI 42 to 59; p<0·0001). Efficacy of dihydroartemisinin-piperaquine plus mefloquine in the three sites in Myanmar was 91% (42 of 46; 95% CI 79 to 98) versus 100% (42 of 42; 95% CI 92 to 100) after dihydroartemisinin-piperaquine (risk difference 9%, 95% CI 1 to 17; p=0·12). The 42-day PCR corrected efficacy of dihydroartemisinin-piperaquine plus mefloquine (96% [68 of 71; 95% CI 88 to 99]) was non-inferior to that of artesunate-mefloquine (95% [69 of 73; 95% CI 87 to 99]) in three sites in Cambodia (risk difference 1%; 95% CI -6 to 8; p=1·00). The overall 42-day PCR-corrected efficacy of artemether-lumefantrine plus amodiaquine (98% [281 of 286; 95% CI 97 to 99]) was similar to that of artemether-lumefantrine (97% [279 of 289; 95% CI 94 to 98]; risk difference 2%, 95% CI -1 to 4; p=0·30). Both TACTs were well tolerated, although early vomiting (within 1 h) was more frequent after dihydroartemisinin-piperaquine plus mefloquine (30 [3·8%] of 794) than after dihydroartemisinin-piperaquine (eight [1·5%] of 543; p=0·012). Vomiting after artemether-lumefantrine plus amodiaquine (22 [1·3%] of 1703) and artemether-lumefantrine (11 [0·6%] of 1721) was infrequent. Adding amodiaquine to artemether-lumefantrine extended the electrocardiogram corrected QT interval (mean increase at 52 h compared with baseline of 8·8 ms [SD 18·6] vs 0·9 ms [16·1]; p<0·01) but adding mefloquine to dihydroartemisinin-piperaquine did not (mean increase of 22·1 ms [SD 19·2] for dihydroartemisinin-piperaquine vs 20·8 ms [SD 17·8] for dihydroartemisinin-piperaquine plus mefloquine; p=0·50).Interpretation: Dihydroartemisinin-piperaquine plus mefloquine and artemether-lumefantrine plus amodiaquine TACTs are efficacious, well tolerated, and safe treatments of uncomplicated P falciparum malaria, including in areas with artemisinin and ACT partner-drug resistance.Funding: UK Department for International Development, Wellcome Trust, Bill & Melinda Gates Foundation, UK Medical Research Council, and US National Institutes of Health. [ABSTRACT FROM AUTHOR]