Genetic complexity alters drug susceptibility of asexual and gametocyte stages of Plasmodium falciparum to antimalarial candidates.

Malaria elimination requires interventions able to target both the asexual blood stage (ABS) parasites and transmissible gametocyte stages of Plasmodium falciparum. Lead antimalarial candidates are evaluated against clinical isolates to address key concerns regarding efficacy and to confirm that the current, circulating parasites from endemic regions lack resistance against these candidates. While this has largely been performed on ABS parasites, limited data are available on the transmission-blocking efficacy of compounds with multistage activity. Here, we evaluated the efficacy of lead antimalarial candidates against both ABS parasites and late-stage gametocytes side-by-side, against clinical P. falciparum isolates from southern Africa. We additionally correlated drug efficacy to the genetic diversity of the clinical isolates as determined with a panel of well-characterized, genome-spanning microsatellite markers. Our data indicate varying sensitivities of the isolates to key antimalarial candidates, both for ABS parasites and gametocyte stages. While ABS parasites were efficiently killed, irrespective of genetic complexity, antimalarial candidates lost some gametocytocidal efficacy when the gametocytes originated from genetically complex, multiple-clone infections. This suggests a fitness benefit to multiclone isolates to sustain transmission and reduce drug susceptibility. In conclusion, this is the first study to investigate the efficacy of antimalarial candidates on both ABS parasites and gametocytes from P. falciparum clinical isolates where the influence of parasite genetic complexity is highlighted, ultimately aiding the malaria elimination agenda.

Parasite genetic diversity reflects continued residual malaria transmission in Vhembe District, a hotspot in the Limpopo Province of South Africa.

BACKGROUND: South Africa aims to eliminate malaria transmission by 2023. However, despite sustained vector control efforts and case management interventions, the Vhembe District remains a malaria transmission hotspot. To better understand Plasmodium falciparum transmission dynamics in the area, this study characterized the genetic diversity of parasites circulating within the Vhembe District. METHODS: A total of 1153 falciparum-positive rapid diagnostic tests (RDTs) were randomly collected from seven clinics within the district, over three consecutive years (2016, 2017 and 2018) during the wet and dry malaria transmission seasons. Using 26 neutral microsatellite markers, differences in genetic diversity were described using a multiparameter scale of multiplicity of infection (MOI), inbreeding metric (Fws), number of unique alleles (A), expected heterozygosity (He), multilocus linkage disequilibrium (LD) and genetic differentiation, and were associated with temporal and geospatial variances. RESULTS: A total of 747 (65%) samples were successfully genotyped. Moderate to high genetic diversity (mean He = 0.74 ± 0.03) was observed in the parasite population. This was ascribed to high allelic richness (mean A = 12.2 ± 1.2). The majority of samples (99%) had unique multi-locus genotypes, indicating high genetic diversity in the sample set. Complex infections were observed in 66% of samples (mean MOI = 2.13 ± 0.04), with 33% of infections showing high within-host diversity as described by the Fws metric. Low, but significant LD (standardised index of association, ISA = 0.08, P < 0.001) was observed that indicates recombination of distinct clones. Limited impact of temporal (FST range - 0.00005 to 0.0003) and spatial (FST = - 0.028 to 0.023) variation on genetic diversity existed during the sampling timeframe and study sites respectively. CONCLUSIONS: Consistent with the Vhembe District's classification as a 'high' transmission setting within South Africa, P. falciparum diversity in the area was moderate to high and complex. This study showed that genetic diversity within the parasite population reflects the continued residual transmission observed in the Vhembe District. This data can be used as a reference point for the assessment of the effectiveness of on-going interventions over time, the identification of imported cases and/or outbreaks, as well as monitoring for the potential spread of anti-malarial drug resistance.

Sensitive and modular amplicon sequencing of Plasmodium falciparum diversity and resistance for research and public health.

Targeted amplicon sequencing is a powerful and efficient tool to interrogate the P. falciparum genome and generate actionable data from infections to complement traditional malaria epidemiology. For maximum impact, genomic tools should be multi-purpose, robust, sensitive and reproducible. We developed, characterized, and implemented MAD4HatTeR, an amplicon sequencing panel based on Multiplex Amplicons for Drug, Diagnostic, Diversity, and Differentiation Haplotypes using Targeted Resequencing, along with a bioinformatic pipeline for data analysis. MAD4HatTeR targets 165 highly diverse loci, focusing on multiallelic microhaplotypes; key markers for drug and diagnostic resistance, including duplications and deletions; and csp and potential vaccine targets. In addition, it can detect non-falciparum Plasmodium species. We used laboratory control and field sample data to demonstrate the high sensitivity and robustness of the panel. The successful implementation of this method in five laboratories, including three in malaria-endemic African countries, showcases its feasibility in generating reproducible data across laboratories. Finally, we introduce an analytical approach to detect gene duplications and deletions from amplicon sequencing data. MAD4HatTeR is thus a powerful research tool and a robust resource for malaria public health surveillance and control.

Population structure and genetic connectivity of Plasmodium falciparum in pre-elimination settings of Southern Africa.

To accelerate malaria elimination in the Southern African region by 2030, it is essential to prevent cross-border malaria transmission. However, countries within the region are highly interconnected due to human migration that aids in the movement of the parasite across geographical borders. It is therefore important to better understand Plasmodium falciparum transmission dynamics in the region, and identify major parasite source and sink populations, as well as cross-border blocks of high parasite connectivity. We performed a meta-analysis using collated parasite allelic data generated by microsatellite genotyping of malaria parasites from Namibia, Eswatini, South Africa, and Mozambique (N = 5,314). The overall number of unique alleles was significantly higher (P ≤ 0.01) in Namibia (mean A = 17.3 ± 1.46) compared to South Africa (mean A = 12.2 ± 1.22) and Eswatini (mean A = 13.3 ± 1.27, P ≤ 0.05), whilst the level of heterozygosity was not significantly different between countries. The proportion of polyclonal infections was highest for Namibia (77%), and lowest for Mozambique (64%). A was significant population structure was detected between parasites from the four countries, and patterns of gene flow showed that Mozambique was the major source area and Eswatini the major sink area of parasites between the countries. This study showed strong signals of parasite population structure and genetic connectivity between malaria parasite populations across national borders. This calls for strengthening the harmonization of malaria control and elimination efforts between countries in the southern African region. This data also proves its potential utility as an additional surveillance tool for malaria surveillance on both a national and regional level for the identification of imported cases and/or outbreaks, as well as monitoring for the potential spread of anti-malarial drug resistance as countries work towards malaria elimination.