Temporal and spatial dynamics of Plasmodium falciparum clonal lineages in Guyana.

Plasmodium parasites, the causal agents of malaria, are eukaryotic organisms that obligately undergo sexual recombination within mosquitoes. In low transmission settings, parasites recombine with themselves, and the clonal lineage is propagated rather than broken up by outcrossing. We investigated whether stochastic/neutral factors drive the persistence and abundance of Plasmodium falciparum clonal lineages in Guyana, a country with relatively low malaria transmission, but the only setting in the Americas in which an important artemisinin resistance mutation (pfk13 C580Y) has been observed. We performed whole genome sequencing on 1,727 Plasmodium falciparum samples collected from infected patients across a five-year period (2016-2021). We characterized the relatedness between each pair of monoclonal infections (n = 1,409) through estimation of identity-by-descent (IBD) and also typed each sample for known or candidate drug resistance mutations. A total of 160 multi-isolate clones (mean IBD ≥ 0.90) were circulating in Guyana during the study period, comprising 13 highly related clusters (mean IBD ≥ 0.40). In the five-year study period, we observed a decrease in frequency of a mutation associated with artemisinin partner drug (piperaquine) resistance (pfcrt C350R) and limited co-occurence of pfcrt C350R with duplications of plasmepsin 2/3, an epistatic interaction associated with piperaquine resistance. We additionally observed 61 nonsynonymous substitutions that increased markedly in frequency over the study period as well as a novel pfk13 mutation (G718S). However, P. falciparum clonal dynamics in Guyana appear to be largely driven by stochastic factors, in contrast to other geographic regions, given that clones carrying drug resistance polymorphisms do not demonstrate enhanced persistence or higher abundance than clones carrying polymorphisms of comparable frequency that are unrelated to resistance. The use of multiple artemisinin combination therapies in Guyana may have contributed to the disappearance of the pfk13 C580Y mutation.

Temporal and spatial dynamics of Plasmodium falciparum clonal lineages in Guyana.

Plasmodium parasites, the causal agents of malaria, are eukaryotic organisms that obligately undergo sexual recombination within mosquitoes. However, in low transmission settings where most mosquitoes become infected with only a single parasite clone, parasites recombine with themselves, and the clonal lineage is propagated rather than broken up by outcrossing. We investigated whether stochastic/neutral factors drive the persistence and abundance of Plasmodium falciparum clonal lineages in Guyana, a country with relatively low malaria transmission, but the only setting in the Americas in which an important artemisinin resistance mutation (pfk13 C580Y) has been observed. To investigate whether this clonality was potentially associated with the persistence and spatial spread of the mutation, we performed whole genome sequencing on 1,727 Plasmodium falciparum samples collected from infected patients across a five-year period (2016-2021). We characterized the relatedness between each pair of monoclonal infections (n=1,409) through estimation of identity by descent (IBD) and also typed each sample for known or candidate drug resistance mutations. A total of 160 clones (mean IBD ≥ 0.90) were circulating in Guyana during the study period, comprising 13 highly related clusters (mean IBD ≥ 0.40). In the five-year study period, we observed a decrease in frequency of a mutation associated with artemisinin partner drug (piperaquine) resistance (pfcrt C350R) and limited co-occurence of pfcrt C350R with duplications of plasmepsin 2/3, an epistatic interaction associated with piperaquine resistance. We additionally report polymorphisms exhibiting evidence of selection for drug resistance or other phenotypes and reported a novel pfk13 mutation (G718S) as well as 61 nonsynonymous substitutions that increased markedly in frequency. However, P. falciparum clonal dynamics in Guyana appear to be largely driven by stochastic factors, in contrast to other geographic regions. The use of multiple artemisinin combination therapies in Guyana may have contributed to the disappearance of the pfk13 C580Y mutation.

Design and implementation of multiplexed amplicon sequencing panels to serve genomic epidemiology of infectious disease: A malaria case study.

Multiplexed PCR amplicon sequencing (AmpSeq) is an increasingly popular application for cost-effective monitoring of threatened species and managed wildlife populations, and shows strong potential for the genomic epidemiology of infectious disease. AmpSeq data from infectious microbes can inform disease control in multiple ways, such as by measuring drug resistance marker prevalence, distinguishing imported from local cases, and determining the effectiveness of therapeutics. We describe the design and comparative evaluation of two new AmpSeq assays for Plasmodium falciparum malaria parasites: a four-locus panel ("4CAST") composed of highly diverse antigens, and a 129-locus panel ("AMPLseq") composed of drug resistance markers, highly diverse loci for inferring relatedness, and a locus to detect Plasmodium vivax co-infection. We explore the performance of each panel in various public health use cases with in silico simulations as well as empirical experiments. The 4CAST panel appears highly suitable for evaluating the number of distinct parasite strains within samples (complexity of infection), showing strong performance across a wide range of parasitaemia levels without a DNA pre-amplification step. For relatedness inference, the larger AMPLseq panel performs similarly to two existing panels of comparable size, despite differences in the data and approach used for designing each panel. Finally, we describe an R package (paneljudge) that facilitates the design and comparative evaluation of genetic panels for relatedness estimation, and we provide general guidance on the design and implementation of AmpSeq panels for the genomic epidemiology of infectious disease.

Malaria care-seeking and treatment ideation among gold miners in Guyana.

BACKGROUND: Although miners are a priority population in malaria elimination in Guyana, scant literature exists on the drivers of malaria-related behaviour. This study explores the relationship between gold miners' malaria-related ideation and the adoption of malaria care-seeking and treatment behaviours including prompt care-seeking, malaria testing, and self-medication. METHODS: Data are from a cross-sectional quantitative survey of 1685 adult miners between the ages of 18-59 years who live in mining camps in Regions 1, 7, and 8. The analysis focused on miners who reported an episode of fever in the past year (n = 745). Malaria care-seeking and treatment ideation was defined as a composite additive score consisting of the following variables: general malaria knowledge, perceived severity, perceived susceptibility, beliefs, perceived self-efficacy, perceived norms, interpersonal communication, and perceived response efficacy. Multivariable logistic regressions explored the relationship between ideation on care-seeking/treatment behaviours, controlling for confounding variables. RESULTS: Most miners with a recent episode of fever had perceived risk (92%), self-efficacy (67%), susceptibility (53%) and high malaria knowledge (53%). Overall, miners' care-seeking/treatment ideation score ranged from 0 to 8 with a mean of 4.1. Ideation scores were associated with higher odds of care-seeking for fever (aOR: 1.19; 95% CI 1.04-1.36), getting tested for malaria (aOR: 1.22; 95% CI 1.07-1.38) and lower odds of self-medication (aOR: 0.87; 95% CI 0.77-0.99). CONCLUSIONS: A national community case management initiative is using study findings as part of its scale-up, using volunteers to make testing and treatment services more accessible to miners. This is complemented by a multi-channel mass media campaign to improve miners' ideation. Communication messages focus on increasing miners' knowledge of malaria transmission and symptoms, encourage positive beliefs about malaria testing and volunteer testers, promote evidence about the effectiveness of testing, and reminders of how quick and easy it is to get a malaria test with the community case management initiative. Study findings also have implications for efforts to eliminate malaria across the Guiana Shield.