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Communications Biology May 2024The Anopheles gambiae 1000 Genomes (Ag1000G) Consortium previously utilized deep sequencing methods to catalogue genetic diversity across African An. gambiae...
The Anopheles gambiae 1000 Genomes (Ag1000G) Consortium previously utilized deep sequencing methods to catalogue genetic diversity across African An. gambiae populations. We analyzed the complete datasets of 1142 individually sequenced mosquitoes through Microsoft Premonition's Bayesian mixture model based (BMM) metagenomics pipeline. All specimens were confirmed as either An. gambiae sensu stricto (s.s.) or An. coluzzii with a high degree of confidence ( > 98% identity to reference). Homo sapiens DNA was identified in all specimens indicating contamination may have occurred either at the time of specimen collection, preparation and/or sequencing. We found evidence of vertebrate hosts in 162 specimens. 59 specimens contained validated Plasmodium falciparum reads. Human hepatitis B and primate erythroparvovirus-1 viral sequences were identified in fifteen and three mosquito specimens, respectively. 478 of the 1,142 specimens were found to contain bacterial reads and bacteriophage-related contigs were detected in 27 specimens. This analysis demonstrates the capacity of metagenomic approaches to elucidate important vector-host-pathogen interactions of epidemiological significance.
Topics: Animals; Anopheles; Metagenomics; Genome, Insect; Mosquito Vectors; Humans; Genetic Variation; Plasmodium falciparum; Metagenome
PubMed: 38816486
DOI: 10.1038/s42003-024-06337-9 -
Nature Communications May 2024The human infectious reservoir of Plasmodium falciparum is governed by transmission efficiency during vector-human contact and mosquito biting preferences. Understanding...
The human infectious reservoir of Plasmodium falciparum is governed by transmission efficiency during vector-human contact and mosquito biting preferences. Understanding biting bias in a natural setting can help target interventions to interrupt transmission. In a 15-month cohort in western Kenya, we detected P. falciparum in indoor-resting Anopheles and human blood samples by qPCR and matched mosquito bloodmeals to cohort participants using short-tandem repeat genotyping. Using risk factor analyses and discrete choice models, we assessed mosquito biting behavior with respect to parasite transmission. Biting was highly unequal; 20% of people received 86% of bites. Biting rates were higher on males (biting rate ratio (BRR): 1.68; CI: 1.28-2.19), children 5-15 years (BRR: 1.49; CI: 1.13-1.98), and P. falciparum-infected individuals (BRR: 1.25; CI: 1.01-1.55). In aggregate, P. falciparum-infected school-age (5-15 years) boys accounted for 50% of bites potentially leading to onward transmission and had an entomological inoculation rate 6.4x higher than any other group. Additionally, infectious mosquitoes were nearly 3x more likely than non-infectious mosquitoes to bite P. falciparum-infected individuals (relative risk ratio 2.76, 95% CI 1.65-4.61). Thus, persistent P. falciparum transmission was characterized by disproportionate onward transmission from school-age boys and by the preference of infected mosquitoes to feed upon infected people.
Topics: Humans; Anopheles; Animals; Plasmodium falciparum; Malaria, Falciparum; Male; Adolescent; Child; Child, Preschool; Female; Kenya; Mosquito Vectors; Insect Bites and Stings; Adult; Feeding Behavior; Young Adult; Infant
PubMed: 38816383
DOI: 10.1038/s41467-024-49080-9 -
PLOS Global Public Health 2024Molecular epidemiologic studies of malaria parasites and other pathogens commonly employ amplicon deep sequencing (AmpSeq) of marker genes derived from dried blood spots...
Molecular epidemiologic studies of malaria parasites and other pathogens commonly employ amplicon deep sequencing (AmpSeq) of marker genes derived from dried blood spots (DBS) to answer public health questions related to topics such as transmission and drug resistance. As these methods are increasingly employed to inform direct public health action, it is important to rigorously evaluate the risk of false positive and false negative haplotypes derived from clinically-relevant sample types. We performed a control experiment evaluating haplotype recovery from AmpSeq of 5 marker genes (ama1, csp, msp7, sera2, and trap) from DBS containing mixtures of DNA from 1 to 10 known P. falciparum reference strains across 3 parasite densities in triplicate (n = 270 samples). While false positive haplotypes were present across all parasite densities and mixtures, we optimized censoring criteria to remove 83% (148/179) of false positives while removing only 8% (67/859) of true positives. Post-censoring, the median pairwise Jaccard distance between replicates was 0.83. We failed to recover 35% (477/1365) of haplotypes expected to be present in the sample. Haplotypes were more likely to be missed in low-density samples with <1.5 genomes/μL (OR: 3.88, CI: 1.82-8.27, vs. high-density samples with ≥75 genomes/μL) and in samples with lower read depth (OR per 10,000 reads: 0.61, CI: 0.54-0.69). Furthermore, minority haplotypes within a sample were more likely to be missed than dominant haplotypes (OR per 0.01 increase in proportion: 0.96, CI: 0.96-0.97). Finally, in clinical samples the percent concordance across markers for multiplicity of infection ranged from 40%-80%. Taken together, our observations indicate that, with sufficient read depth, the majority of haplotypes can be successfully recovered from DBS while limiting the false positive rate.
PubMed: 38814915
DOI: 10.1371/journal.pgph.0002361 -
International Journal For Parasitology.... Apr 2024Target-based approaches have traditionally been used in the search for new anti-infective molecules. Target selection process, a critical step in Drug Discovery,...
Target-based approaches have traditionally been used in the search for new anti-infective molecules. Target selection process, a critical step in Drug Discovery, identifies targets that are essential to establish or maintain the infection, tractable to be susceptible for inhibition, selective towards their human ortholog and amenable for large scale purification and high throughput screening. The work presented herein validates the Plasmodium falciparum mRNA 5' triphosphatase (PfPRT1), the first enzymatic step to cap parasite nuclear mRNAs, as a candidate target for the development of new antimalarial compounds. mRNA capping is essential to maintain the integrity and stability of the messengers, allowing their translation. PfPRT1 has been identified as a member of the tunnel, metal dependent mRNA 5' triphosphatase family which differs structurally and mechanistically from human metal independent mRNA 5' triphosphatase. In the present study the essentiality of PfPRT1 was confirmed and molecular biology tools and methods for target purification, enzymatic assessment and target engagement were developed, with the goal of running a future high throughput screening to discover PfPRT1 inhibitors.
PubMed: 38810336
DOI: 10.1016/j.ijpddr.2024.100537 -
Frontiers in Cellular and Infection... 2024Diversity in malarial antigens is an immune evasion mechanism that gives malaria parasites an edge over the host. Immune responses against one variant of a polymorphic...
INTRODUCTION
Diversity in malarial antigens is an immune evasion mechanism that gives malaria parasites an edge over the host. Immune responses against one variant of a polymorphic antigen are usually not fully effective against other variants due to altered epitopes. This study aimed to evaluate diversity in the Plasmodium falciparum antigens apical membrane antigen 1 (PfAMA1) and circumsporozoite protein (PfCSP) from circulating parasites in a malaria-endemic community in southern Ghana and to determine the effects of polymorphisms on antibody response specificity.
METHODS
The study involved 300 subjects, whose infection status was determined by microscopy and PCR. Diversity within the two antigens was evaluated by msp2 gene typing and molecular gene sequencing, while the host plasma levels of antibodies against PfAMA1, PfCSP, and two synthetic 24mer peptides from the conserved central repeat region of PfCSP, were measured by ELISA.
RESULTS
Of the 300 subjects, 171 (57%) had infection, with 165 of the 171 (96.5%) being positive for either or both of the allelic families. Gene sequencing of DNA from 55 clonally infected samples identified a total of 56 non-synonymous single nucleotide polymorphisms (SNPs) for the gene and these resulted in 44 polymorphic positions, including two novel positions (363 and 365). Sequencing of the Pfcsp gene from 69 clonal DNA samples identified 50 non-synonymous SNPs that resulted in 42 polymorphic positions, with half (21) of these polymorphic positions being novel. Of the measured antibodies, only anti-PfCSP antibodies varied considerably between PCR parasite-positive and parasite-negative persons.
DISCUSSION
These data confirm the presence of a considerable amount of unique, previously unreported amino acid changes, especially within PfCSP. Drivers for this diversity in the Pfcsp gene do not immediately seem apparent, as immune pressure will be expected to drive a similar level of diversity in the gene.
Topics: Plasmodium falciparum; Antigens, Protozoan; Ghana; Humans; Protozoan Proteins; Malaria, Falciparum; Membrane Proteins; Antibodies, Protozoan; Female; Adult; Male; Adolescent; Young Adult; Child; Genetic Variation; Child, Preschool; Middle Aged; Sequence Analysis, DNA; Enzyme-Linked Immunosorbent Assay; Polymerase Chain Reaction; Antigenic Variation; DNA, Protozoan
PubMed: 38808064
DOI: 10.3389/fcimb.2024.1375249 -
Malaria Journal May 2024Deforestation is an important driver of malaria dynamics, with a relevant impact on mosquito ecology, including larval habitat availability, blood-feeding behaviour, and...
BACKGROUND
Deforestation is an important driver of malaria dynamics, with a relevant impact on mosquito ecology, including larval habitat availability, blood-feeding behaviour, and peak biting time. The latter is one of several entomological metrics to evaluate vectorial capacity and effectiveness of disease control. This study aimed to test the effect of forest cover percentage on the peak biting time of Plasmodium-uninfected and infected Nyssorhynchus darlingi females.
METHODS
Mosquitoes were captured utilizing human landing catch (HLC) in the peridomestic habitat in field collections carried out in the wet, wet-dry transition, and dry seasons from 2014 to 2017 in areas with active malaria transmission in Amazonian Brazil. The study locations were in rural settlements in areas with the mean annual malaria parasite incidence (Annual Parasite Incidence, API ≥ 30). All Ny. darlingi females were tested for Plasmodium spp. infection using real time PCR technique. Forest cover percentage was calculated for each collection site using QGIS v. 2.8 and was categorized in three distinct deforestation scenarios: (1) degraded, < 30% forest cover, (2) intermediate, 30-70% forest cover, and (3) preserved, > 70% forest cover.
RESULTS
The highest number of uninfected female Ny. darlingi was found in degraded landscape-sites with forest cover < 30% in any peak biting time between 18:00 and 0:00. Partially degraded landscape-sites, with (30-70%) forest cover, showed the highest number of vivax-infected females, with a peak biting time of 21:00-23:00. The number of P. falciparum-infected mosquitoes was highest in preserved sites with > 70% forest cover, a peak biting at 19:00-20:00, and in sites with 30-70% forest cover at 22:00-23:00.
CONCLUSIONS
Results of this study show empirically that degraded landscapes favour uninfected Ny. darlingi with a peak biting time at dusk (18:00-19:00), whereas partially degraded landscapes affect the behaviour of Plasmodium-infected Ny. darlingi by shifting its peak biting time towards hours after dark (21:00-23:00). In preserved sites, Plasmodium-infected Ny. darlingi bite around dusk (18:00-19:00) and shortly after (19:00-20:00).
Topics: Animals; Brazil; Forests; Female; Feeding Behavior; Mosquito Vectors; Conservation of Natural Resources; Insect Bites and Stings; Seasons; Malaria
PubMed: 38807105
DOI: 10.1186/s12936-024-04984-1 -
International Journal For Parasitology.... May 2024Plasmodium falciparum aminoacyl tRNA synthetases (PfaaRSs) are potent antimalarial targets essential for proteome fidelity and overall parasite survival in every stage...
Plasmodium falciparum aminoacyl tRNA synthetases (PfaaRSs) are potent antimalarial targets essential for proteome fidelity and overall parasite survival in every stage of the parasite's life cycle. So far, some of these proteins have been singly targeted yielding inhibitor compounds that have been limited by incidences of resistance which can be overcome via pan-inhibition strategies. Hence, herein, for the first time, we report the identification and in vitro antiplasmodial validation of Mitomycin (MMC) as a probable pan-inhibitor of class 1a (arginyl(A)-, cysteinyl(C), isoleucyl(I)-, leucyl(L), methionyl(M), and valyl(V)-) PfaaRSs which hypothetically may underlie its previously reported activity on the ribosomal RNA to inhibit protein translation and biosynthesis. We combined multiple in silico structure-based discovery strategies that first helped identify functional and druggable sites that were preferentially targeted by the compound in each of the plasmodial proteins: Ins1-Ins2 domain in Pf-ARS; anticodon binding domain in Pf-CRS; CP1-editing domain in Pf-IRS and Pf-MRS; C-terminal domain in Pf-LRS; and CP-core region in Pf-VRS. Molecular dynamics studies further revealed that MMC allosterically induced changes in the global structures of each protein. Likewise, prominent structural perturbations were caused by the compound across the functional domains of the proteins. More so, MMC induced systematic alterations in the binding of the catalytic nucleotide and amino acid substrates which culminated in the loss of key interactions with key active site residues and ultimate reduction in the nucleotide-binding affinities across all proteins, as deduced from the binding energy calculations. These altogether confirmed that MMC uniformly disrupted the structure of the target proteins and essential substrates. Further, MMC demonstrated IC < 5 μM against the Dd2 and 3D7 strains of parasite making it a good starting point for malarial drug development. We believe that findings from our study will be important in the current search for highly effective multi-stage antimalarial drugs.
PubMed: 38805932
DOI: 10.1016/j.ijpddr.2024.100548 -
Life Science Alliance Aug 2024The merozoite surface protein 1 (MSP1) is the most abundant protein on the surface of the invasive merozoite stages of and has long been considered a key target of...
The merozoite surface protein 1 (MSP1) is the most abundant protein on the surface of the invasive merozoite stages of and has long been considered a key target of protective immunity. We used samples from a single controlled human malaria challenge study to test whether the full-length version of MSP1 (MSP1) induced antibodies that mediated Fc-IgG functional activity in five independent assays. We found that anti-MSP1 antibodies induced complement fixation via C1q, monocyte-mediated phagocytosis, neutrophil respiratory burst, and natural killer cell degranulation as well as IFNγ production. Activity in each of these assays was strongly associated with protection. The breadth of MSP1-specific Fc-mediated effector functions was more strongly associated with protection than the individual measures and closely mirrored what we have previously reported using the same assays against merozoites. Our findings suggest that MSP1 is an important target of functional antibodies that contribute to a protective immune response against malaria.
Topics: Humans; Merozoite Surface Protein 1; Malaria, Falciparum; Plasmodium falciparum; Antibodies, Protozoan; Phagocytosis; Immunoglobulin G; Killer Cells, Natural; Interferon-gamma; Female; Merozoites; Neutrophils
PubMed: 38803222
DOI: 10.26508/lsa.202301910 -
Scientific Reports May 2024Field-derived metrics are critical for effective control of malaria, particularly in sub-Saharan Africa where the disease kills over half a million people yearly. One...
Field-derived metrics are critical for effective control of malaria, particularly in sub-Saharan Africa where the disease kills over half a million people yearly. One key metric is entomological inoculation rate, a direct measure of transmission intensities, computed as a product of human biting rates and prevalence of Plasmodium sporozoites in mosquitoes. Unfortunately, current methods for identifying infectious mosquitoes are laborious, time-consuming, and may require expensive reagents that are not always readily available. Here, we demonstrate the first field-application of mid-infrared spectroscopy and machine learning (MIRS-ML) to swiftly and accurately detect Plasmodium falciparum sporozoites in wild-caught Anopheles funestus, a major Afro-tropical malaria vector, without requiring any laboratory reagents. We collected 7178 female An. funestus from rural Tanzanian households using CDC-light traps, then desiccated and scanned their heads and thoraces using an FT-IR spectrometer. The sporozoite infections were confirmed using enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR), to establish references for training supervised algorithms. The XGBoost model was used to detect sporozoite-infectious specimen, accurately predicting ELISA and PCR outcomes with 92% and 93% accuracies respectively. These findings suggest that MIRS-ML can rapidly detect P. falciparum in field-collected mosquitoes, with potential for enhancing surveillance in malaria-endemic regions. The technique is both fast, scanning 60-100 mosquitoes per hour, and cost-efficient, requiring no biochemical reactions and therefore no reagents. Given its previously proven capability in monitoring key entomological indicators like mosquito age, human blood index, and identities of vector species, we conclude that MIRS-ML could constitute a low-cost multi-functional toolkit for monitoring malaria risk and evaluating interventions.
Topics: Animals; Anopheles; Malaria, Falciparum; Plasmodium falciparum; Machine Learning; Mosquito Vectors; Female; Humans; Tanzania; Sporozoites; Spectrophotometry, Infrared; Spectroscopy, Fourier Transform Infrared
PubMed: 38802488
DOI: 10.1038/s41598-024-63082-z -
BioRxiv : the Preprint Server For... Jun 2024malaria parasites invade and multiply inside red blood cells (RBCs), the most iron-rich compartment in humans. Like all cells, requires nutritional iron to support...
malaria parasites invade and multiply inside red blood cells (RBCs), the most iron-rich compartment in humans. Like all cells, requires nutritional iron to support essential metabolic pathways, but the critical mechanisms of iron acquisition and trafficking during RBC infection have remained obscure. Parasites internalize and liberate massive amounts of heme during large-scale digestion of RBC hemoglobin within an acidic food vacuole (FV) but lack a heme oxygenase to release porphyrin-bound iron. Although most FV heme is sequestered into inert hemozoin crystals, prior studies indicate that trace heme escapes biomineralization and is susceptible to non-enzymatic degradation within the oxidizing FV environment to release labile iron. Parasites retain a homolog of divalent metal transporter 1 (DMT1), a known mammalian iron transporter, but its role in iron acquisition has not been tested. Our phylogenetic studies indicate that DMT1 (PfDMT1) retains conserved molecular features critical for metal transport. We localized this protein to the FV membrane and defined its orientation in an export-competent topology. Conditional knockdown of PfDMT1 expression is lethal to parasites, which display broad cellular defects in iron-dependent functions, including impaired apicoplast biogenesis and mitochondrial polarization. Parasites are selectively rescued from partial PfDMT1 knockdown by supplementation with exogenous iron, but not other metals. These results support a cellular paradigm whereby PfDMT1 is the molecular gatekeeper to essential iron acquisition by blood-stage malaria parasites and suggest that therapeutic targeting of PfDMT1 may be a potent antimalarial strategy.
PubMed: 38798484
DOI: 10.1101/2024.05.10.587216