Biology subjects

Conrad, M. D.

Publications and source records attributed to Conrad, M. D..

5 recordsLinked to original sources

bioRxiv2026

Whole-genome characterization of Plasmodium falciparum population structure and drug resistance across Uganda and neighboring countries

Uganda has one of the world's highest malaria burdens, but parasite population structure and connectivity have not been characterized nationally with whole-genome data. We analyzed 249 monoclonal Plasmodium falciparum genomes from 26 sentinel sites across Uganda in 2023 and compared them with publicly available genomes from five neighboring countries. Genetic diversity was high and differentiation low in Uganda, but identity-by-descent detected limited structure. This was most pronounced in the low-transmission southwest, where parasites shared ancestry and recent relatedness with isolates from western Tanzania. Selection scans identified signals at loci associated with resistance to artemisinins and their partner drugs, and to chloroquine and antifolates. The strongest signal was at PfPX1 where the PIN haplotype (L1222P, M1701I, D1705N) reached 70% prevalence nationwide, but selection was carried by PIN haplotypes that had additionally acquired D384A, largely confined to northern and eastern Uganda. A second substitution at the same codon, D384Y, formed a haplotype shared across the Tanzanian border. PfKelch13 variants were partitioned across multiple backgrounds, consistent with repeated independent emergence, and C469Y was enriched on the PfPX1 D384A-PIN background. These genomes provide a 2023 baseline for tracking potential mediators of drug resistance and support coordinated genomic surveillance across Uganda and its neighbors.

genomics↗

bioRxiv2026

PfPHAST: Plasmodium falciparum Public Health Amplicon Sequencing Tool, a Streamlined Panel for Malaria Genomic Surveillance

Genomic tools can support malaria control policy through surveillance of Plasmodium falciparum populations, tracking antimalarial drug resistance, pfhrp2/3 deletions that compromise rapid diagnostic tests, and selection at the circumsporozoite protein (PfCSP) vaccine target, as well as through molecular correction of therapeutic efficacy studies (TES). Multiplex Amplicons for Drug, Diagnostic, Diversity, and Differentiation Haplotypes using Targeted Resequencing (MAD4HatTeR), a comprehensive amplicon sequencing panel covering up to 276 targets, supports these applications but is tailored to research rather than routine programmatic use. We developed P. falciparum Public Health Amplicon Sequencing Tool (PfPHAST), a 56-target derivative of MAD4HatTeR spanning drug resistance loci, pfhrp2/3 deletion, PfCSP genotyping, non-falciparum species identification, and 20 high-heterozygosity microhaplotype loci for TES classification. We compared PfPHAST and MAD4HatTeR using laboratory strain controls, including two-strain dilution series and a five-strain mixture, across parasite densities of 100 to 10,000 parasites/L. At matched per-target depth, PfPHAST achieved a higher quality-control pass rate than MAD4HatTeR (94.4% versus 90.0%) and distributed reads more evenly across targets. The panels showed comparable recall and precision for drug resistance codons and microhaplotypes, reaching near-complete recall above 40% within-sample allele frequency (WSAF) at all densities, with reduced sensitivity for minor alleles below 10% WSAF at low parasite density in both panels. Observed and expected WSAF correlated strongly for both panels, and both resolved a five-strain polyclonal mixture, including a 5% minor strain. By concentrating sequencing capacity on targets of greatest programmatic relevance, PfPHAST offers a scalable, lower-cost alternative to comprehensive research panels without sacrificing performance on shared targets, complementing MAD4HatTeR for routine molecular malaria surveillance.

genomics↗

bioRxiv2025

A novel locus associated with decreased susceptibility of Plasmodium falciparum to lumefantrine and dihydroartemisinin has emerged and spread in Uganda

Malaria control in Uganda is threatened by the emergence of artemisinin partial resistance and reduced lumefantrine susceptibility. To identify loci contributing to decreased drug susceptibility, we assessed signatures of selection in Ugandan whole-genome Plasmodium falciparum sequences. Extended shared haplotypes were seen for Kelch13 C469Y and A675V mutations, but the strongest signal of recent selection was centered on a segment of chromosome 7 encoding the phosphoinositide-binding protein (PX1, PF3D7_0720700). A haplotype, represented by three PX1 mutations (L1222P, M1701I, D1705N) and two deletions (designated PIN), was first seen in 2008 and rapidly increased, reaching prevalence >50% in northern Uganda by 2016 and eastern Uganda by 2023. PIN-carrying parasites showed significantly decreased ex vivo susceptibilities to lumefantrine, mefloquine and dihydroartemisinin. A parasite strain in which px1 was disrupted in vitro showed increased susceptibility to the three drugs. Thus, PX1 polymorphisms appear to impact on the susceptibilities of African malaria parasites to key drugs.

genomics↗

bioRxiv2025

Significant variations in tolerance to clothianidin and pirimiphos-methyl in Anopheles gambiae and Anopheles funestus populations during a dramatic malaria resurgence despite sustained indoor residual spraying in Uganda

A dramatic malaria resurgence occurred in areas of Uganda between 2020 and 2022 coincident with the switch to clothianidin-based formulations for indoor residual spraying. During the resurgence, Anopheles funestus numbers increased but when an alternative insecticide, pirimiphos methyl, was reintroduced in 2023, both malaria cases and An. funestus mosquito density fell. In this study, we investigated possible causes of the resurgence by assessing; 1) whether sufficient quantities of insecticide were sprayed; 2) the residual insecticide bioefficacy against wild mosquitoes and; 3) the insecticide susceptibility of vector populations using standard test tube assays and wall cone assays. In 2023, after adjusting for extraction efficiency, 70-80% of the houses had optimal residual concentrations of insecticides (clothianidin >0.3g/m2; pirimiphos methyl >0.5g/m2) with significant variations between sampling rounds and wall types. Mud walls had the lowest residual concentration of insecticides, and the lowest observed mortality in wall cone assays, compared to fired bricks with plaster/cement/paint. In the studies of residual bio efficacy, by World Health Organization (WHO) definitions, An. funestus showed resistance to clothianidin (<80% mortality) up to 11 months and susceptibility to pirimiphos methyl (>90% mortality) when exposed to wall surfaces up to 7 months post-spray. In WHO tube tests, variations were observed in susceptibility to clothianidin in An. funestus populations using dose- and time-response assays (80-98% mortality). In 2022, An. gambiae was largely susceptible to the clothianidin-based formulation Sumishield (85-90% mortality) although the levels dropped slightly in 2023 (60-85% mortality) mainly in mud and pole houses. In contrast, An. gambiae was mildly susceptible to the pirimiphos methyl-based formulation Actellic ([~]80% mortality) and time response assays showed An. gambiae populations had very low knockdown and mortality at lower exposure time compared to An. funestus. Regression models showed a positive association between residual insecticide concentration (RIC) and mortality in houses sprayed with Sumishield but not Actellic houses. Despite the possible variations observed in spray operations, the study revealed that An. funestus exhibited a higher tolerance to clothianidin-based formulations compared to An. gambiae, and this might have driven the malaria resurgence observed in Uganda. However, there are signals of An. gambiae resistance to pirimiphos-methyl which will require further investigation and monitoring.

zoology↗

bioRxiv2024

Gametocyte production and transmission fitness of African and Asian Plasmodium falciparum isolates with differential susceptibility to artemisinins

The emergence of Plasmodium falciparum parasites partially resistant to artemisinins (ART-R) poses a significant threat to recent gains in malaria control. ART-R has been associated with PfKelch13 (K13) mutations, which differ in fitness costs. This study investigates the gametocyte production and transmission fitness of African and Asian P. falciparum isolates with different K13 genotypes across multiple mosquito species. We tested three ART-sensitive (ART-S) isolates (NF54, NF135, NF180) and three ART-R isolates (ARN1G, 3815, PAT-023) for sexual conversion and transmission to Anopheles stephensi, An. gambiae and An. coluzzii. ART-R levels were quantified in vitro using the Ring-stage Survival Assay (RSA), and the transmission-reducing effects of dihydroartemisinin (DHA) on mature gametocytes were assessed. Results showed that ART-S parasite lines consistently produced gametocytes and transmitted effectively in all three mosquito species. ART-R isolates showed variability: ARN1G maintained high transmission levels, whereas 3815 showed limited transmission potential despite higher sporozoite loads in An. coluzzii. The African ART-R isolate PAT-023 demonstrated low gametocyte commitment but was transmitted efficiently in both An. gambiae and An. coluzzii. DHA exposure reduced mosquito infectivity for all isolates, regardless of K13 genotype. These findings, based on a limited number of field isolates, suggest that ART-R parasites remain transmissible across different Anopheles species. However, ART-R does not appear to confer a direct transmission advantage. This study highlights the complexity of ART-R dynamics and underscores the need for further research to inform malaria control strategies in regions where ART-R parasites are circulating.

microbiology↗