RESUMEN
Malaria-related mortality has slowly decreased over the past decade; however, eradication of malaria requires the development of new antimalarial chemotherapies that target liver stages of the parasite and combat the emergence of drug resistance. The diminishing arsenal of anti-liver-stage compounds sparked our interest in reviving the old and previously abandoned compound menoctone. In support of these studies, we developed a new convergent synthesis method that was facile, required fewer steps, produced better yields, and utilized less expensive reagents than the previously published method. Menoctone proved to be highly potent against liver stages of Plasmodium berghei (50 percent inhibitory concentration [IC50] = 0.41 nM) and erythrocytic stages of Plasmodium falciparum (113 nM). We selected for resistance to menoctone and found M133I mutations in cytochrome b of both P. falciparum and P. berghei The same mutation has been observed previously in atovaquone resistance, and we confirmed cross-resistance between menoctone and atovaquone in vitro (for P. falciparum) and in vivo (for P. berghei). Finally, we assessed the transmission potential of menoctone-resistant P. berghei and found that the M133I mutant parasites were readily transmitted from mouse to mosquitoes and back to mice. In each step, the M133I mutation in cytochrome b, inducing menoctone resistance, was confirmed. In summary, this study is the first to show the mechanism of resistance to menoctone and that menoctone and atovaquone resistance is transmissible through mosquitoes.
Asunto(s)
Anopheles/parasitología , Antimaláricos/farmacología , Atovacuona/farmacología , Citocromos b/genética , Resistencia a Medicamentos/genética , Naftoquinonas/farmacología , Plasmodium berghei/genética , Plasmodium falciparum/genética , Animales , ADN Protozoario/genética , Femenino , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Malaria Falciparum/transmisión , Ratones , Ratones Endogámicos BALB C , Mutación/genética , Plasmodium berghei/efectos de los fármacos , Plasmodium falciparum/efectos de los fármacosRESUMEN
Challenges in classifying recurrent Plasmodium vivax infections constrain surveillance of antimalarial efficacy and transmission. Recurrent infections may arise from activation of dormant liver stages (relapse), blood-stage treatment failure (recrudescence) or reinfection. Molecular inference of familial relatedness (identity-by-descent or IBD) can help resolve the probable origin of recurrences. As whole genome sequencing of P. vivax remains challenging, targeted genotyping methods are needed for scalability. We describe a P. vivax marker discovery framework to identify and select panels of microhaplotypes (multi-allelic markers within small, amplifiable segments of the genome) that can accurately capture IBD. We evaluate panels of 50-250 microhaplotypes discovered in a global set of 615 P. vivax genomes. A candidate global 100-microhaplotype panel exhibits high marker diversity in the Asia-Pacific, Latin America and horn of Africa (median HE = 0.70-0.81) and identifies 89% of the polyclonal infections detected with genome-wide datasets. Data simulations reveal lower error in estimating pairwise IBD using microhaplotypes relative to traditional biallelic SNP barcodes. The candidate global panel also exhibits high accuracy in predicting geographic origin and captures local infection outbreak and bottlenecking events. Our framework is open-source enabling customised microhaplotype discovery and selection, with potential for porting to other species or data resources.
Asunto(s)
Malaria Vivax , Plasmodium vivax , Recurrencia , Plasmodium vivax/genética , Malaria Vivax/parasitología , Malaria Vivax/epidemiología , Humanos , Haplotipos/genética , Polimorfismo de Nucleótido Simple , Genoma de Protozoos/genética , GenotipoRESUMEN
Ethiopia has the greatest burden of Plasmodium vivax in Africa, but little is known about the epidemiological landscape of parasites across the country. We analysed the genomic diversity of 137 P. vivax isolates collected nine Ethiopian districts from 2012 to 2016. Signatures of selection were detected by cross-country comparisons with isolates from Thailand (n = 104) and Indonesia (n = 111), representing regions with low and high chloroquine resistance respectively. 26% (35/137) of Ethiopian infections were polyclonal, and 48.5% (17/35) of these comprised highly related clones (within-host identity-by-descent > 25%), indicating frequent co-transmission and superinfection. Parasite gene flow between districts could not be explained entirely by geographic distance, with economic and cultural factors hypothesised to have an impact on connectivity. Amplification of the duffy binding protein gene (pvdbp1) was prevalent across all districts (16-75%). Cross-population haplotype homozygosity revealed positive selection in a region proximal to the putative chloroquine resistance transporter gene (pvcrt-o). An S25P variant in amino acid transporter 1 (pvaat1), whose homologue has recently been implicated in P. falciparum chloroquine resistance evolution, was prevalent in Ethiopia (96%) but not Thailand or Indonesia (35-53%). The genomic architecture in Ethiopia highlights circulating variants of potential public health concern in an endemic setting with evidence of stable transmission.
Asunto(s)
Antimaláricos , Malaria Falciparum , Malaria Vivax , Humanos , Plasmodium vivax , Malaria Vivax/parasitología , Etiopía/epidemiología , Cloroquina/farmacología , Cloroquina/uso terapéutico , Malaria Falciparum/parasitología , Genómica , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Resistencia a Medicamentos/genética , Plasmodium falciparum/metabolismoRESUMEN
Challenges in understanding the origin of recurrent Plasmodium vivax infections constrains the surveillance of antimalarial efficacy and transmission of this neglected parasite. Recurrent infections within an individual may arise from activation of dormant liver stages (relapse), blood-stage treatment failure (recrudescence) or new inoculations (reinfection). Molecular inference of familial relatedness (identity-by-descent or IBD) based on whole genome sequence data, together with analysis of the intervals between parasitaemic episodes ("time-to-event" analysis), can help resolve the probable origin of recurrences. Whole genome sequencing of predominantly low-density P. vivax infections is challenging, so an accurate and scalable genotyping method to determine the origins of recurrent parasitaemia would be of significant benefit. We have developed a P. vivax genome-wide informatics pipeline to select specific microhaplotype panels that can capture IBD within small, amplifiable segments of the genome. Using a global set of 615 P. vivax genomes, we derived a panel of 100 microhaplotypes, each comprising 3-10 high frequency SNPs within <200 bp sequence windows. This panel exhibits high diversity in regions of the Asia-Pacific, Latin America and the horn of Africa (median HE = 0.70-0.81) and it captured 89% (273/307) of the polyclonal infections detected with genome-wide datasets. Using data simulations, we demonstrate lower error in estimating pairwise IBD using microhaplotypes, relative to traditional biallelic SNP barcodes. Our panel exhibited high accuracy in predicting the country of origin (median Matthew's correlation coefficient >0.9 in 90% countries tested) and it also captured local infection outbreak and bottlenecking events. The informatics pipeline is available open-source and yields microhaplotypes that can be readily transferred to high-throughput amplicon sequencing assays for surveillance in malaria-endemic regions.
RESUMEN
Recent genomic studies are investigating the wide-ranging implications of malaria complex infections on parasite diversity, transmission, and downstream disease eradication efforts. Using single cell sequencing, new work by Nkhoma et al. provides evidence that there is unexpectedly frequent co-transmission of related parasites in the intense transmission setting in Malawi.
Asunto(s)
Malaria/parasitología , Plasmodium/clasificación , Plasmodium/genética , Animales , Genoma de Protozoos/genética , Genómica , Interacciones Huésped-Parásitos , Humanos , Malaria/transmisión , Filogenia , Análisis de la Célula IndividualRESUMEN
Plasmodium vivax gene regulation remains difficult to study due to the lack of a robust in vitro culture method, low parasite densities in peripheral circulation and asynchronous parasite development. We adapted an RNA-seq protocol "DAFT-seq" to sequence the transcriptome of four P. vivax field isolates that were cultured for a short period ex vivo before using a density gradient for schizont enrichment. Transcription was detected from 78% of the PvP01 reference genome, despite being schizont-enriched samples. This extensive data was used to define thousands of 5' and 3' untranslated regions, some of which overlapped with neighbouring transcripts, and to improve the gene models of 352 genes, including identifying 20 novel gene transcripts. This dataset has also significantly increased the known amount of heterogeneity between P. vivax schizont transcriptomes from individual patients. The majority of genes found to be differentially expressed between the isolates lack Plasmodium falciparum homologs and are predicted to be involved in host-parasite interactions, with an enrichment in reticulocyte binding proteins, merozoite surface proteins and exported proteins with unknown function. An improved understanding of the diversity within P. vivax transcriptomes will be essential for the prioritisation of novel vaccine targets.
Asunto(s)
Regulación de la Expresión Génica , Interacciones Huésped-Parásitos/genética , Plasmodium vivax/genética , Esquizontes/genética , Transcriptoma , Humanos , Malaria Vivax/parasitología , Merozoítos/genética , Plasmodium vivax/aislamiento & purificación , Esquizontes/aislamiento & purificaciónRESUMEN
Plasmodium species are frequently host-specific, but little is currently known about the molecular factors restricting host switching. This is particularly relevant for P. falciparum, the only known human-infective species of the Laverania sub-genus, all other members of which infect African apes. Here we show that all tested P. falciparum isolates contain an inactivating mutation in an erythrocyte invasion associated gene, PfEBA165, the homologues of which are intact in all ape-infective Laverania species. Recombinant EBA165 proteins only bind ape, not human, erythrocytes, and this specificity is due to differences in erythrocyte surface sialic acids. Correction of PfEBA165 inactivating mutations by genome editing yields viable parasites, but is associated with down regulation of both PfEBA165 and an adjacent invasion ligand, which suggests that PfEBA165 expression is incompatible with parasite growth in human erythrocytes. Pseudogenization of PfEBA165 may represent a key step in the emergence and evolution of P. falciparum.
Asunto(s)
Eritrocitos/parasitología , Especificidad del Huésped/genética , Malaria Falciparum/parasitología , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Animales , Sistemas CRISPR-Cas/genética , Ingeniería Celular , Eritrocitos/metabolismo , Evolución Molecular , Mutación del Sistema de Lectura , Edición Génica , Células HEK293 , Humanos , Mutación con Pérdida de Función , Pan troglodytes/parasitología , Plasmodium falciparum/aislamiento & purificación , Plasmodium falciparum/patogenicidad , Ácidos Siálicos/metabolismoRESUMEN
Life cycles of spirorchiids that infect the vascular system of turtles are poorly understood. Few life cycles of these blood flukes have been elucidated and all intermediate hosts reported are gastropods (Mollusca), regardless of whether the definitive host is a freshwater or a marine turtle. During a recent survey of blood fluke larvae in polychaetes on the coast of South Carolina, USA, spirorchiid-like cercariae were found to infect the polychaetes Amphitrite ornata (Terebellidae) and Enoplobranchus sanguineus (Polycirridae). Cercariae were large, furcate, with a ventral acetabulum, but no eyespots were observed. Partial sequences of D1-D2 domains of the large ribosomal subunit, the internal transcribed spacer 2, and the mitochondrial cytochrome oxidase 1 genes allowed the identification of sporocysts and cercariae as belonging to two unidentified Neospirorchis species reported from the green turtle, Chelonia mydas, in Florida: Neospirorchis sp. (Neogen 13) in A. ornata and Neospirorchis sp. (Neogen 14) in E. sanguineus. Phylogenetic analysis suggests that infection of annelids by blood flukes evolved separately in aporocotylids and spirorchiids. Our results support the contention that the Spirorchiidae is not a valid family and suggest that Neospirorchis is a monophyletic clade within the paraphyletic Spirorchiidae. Since specificity of spirorchiids for their intermediate hosts is broader than it was thus far assumed, surveys of annelids in turtle habitats are necessary to further our understanding of the life history of these pathogenic parasites.
Asunto(s)
Oocistos/aislamiento & purificación , Poliquetos/parasitología , Trematodos/aislamiento & purificación , Tortugas/parasitología , Animales , Interacciones Huésped-ParásitosRESUMEN
Aporocotylidae comprises a diverse family of fish blood flukes, with adults found in blood or body cavity of marine, brackish, or freshwater fish. Aporocotylids are unique among the Digenea with many developing in polychaetes. The life cycle has been elucidated for only a few species that develop in polychaetes from marine/brackish environments and none for western Atlantic aporocotylids. The basis for this study was observations of blood fluke larvae in annelids from South Carolina, USA in 1982 prior to possible usage of molecular tools to specifically identify parasite larvae. Recent description of aporocotylid species and genotyping tools prompted revisiting original collection sites to examine polychaetes and fish as potential hosts. Polycirrid Enoplobranchus sanguineus and terebellids Amphitrite ornata, and Terebella lapidaria revealed infections with aporocotylid larvae. Adult blood flukes were also collected from fish commonly encountered in the same habitat: spotted seatrout (Cynoscion nebulosus), red drum (Sciaenops ocellatus), black drum (Pogonias cromis), and Atlantic croaker (Micropogonias undulatus). Sporocysts containing cercariae were found in individuals of each annelid species. Adult Cardicola parvus were found in spotted seatrout and Atlantic croaker, C. laruei in spotted seatrout, C. currani in red drum, and C. palmeri in black drum. Genotype analysis of ITS-2 and lsrDNA of all forms confirmed conspecific infections by C. parvus in E. sanguineus and A. ornata and C. laruei in T. lapidaria. This is the first description of complete life cycles of aporocotylids in the Western Atlantic and first evidence of cryptic infections of Cy. nebulosus with C. parvus.