Whole-Genome Sequencing to Evaluate the Resistance Landscape Following Antimalarial Treatment Failure With Fosmidomycin-Clindamycin

Guggisberg, Ann M.; Sundararaman, Sesh A.; Lanaspa, Miguel; Moraleda, Cinta; González, Raquel; Mayor, Alfredo; Cisteró, Pau; Hutchinson, David; Kremsner, Peter G.; Hahn, Beatrice H.; Bassat, Quique; Odom, Audrey R.

doi:10.1093/infdis/jiw304

Cited by 31 publications

(36 citation statements)

References 45 publications

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“…The sWGA method efficiently enriches P. falciparum DNA from infected mosquito and mouse tissues, confirming the performance of this approach for enriching parasite DNA from dried blood spots (Guggisberg et al 2016; Oyola et al 2016; Sundararaman et al 2016; Cowell et al 2017). Our results further show that sWGA does not generate bias in allele frequency measurement (Fig.…”

Section: Discussionsupporting

confidence: 54%

“…sWGA uses short 8-12 mer oligonucleotide probes that preferentially bind to the target genome, rather than random hexamers used in normal whole genome amplification. This approach was pioneered by Leichty and Brisson (Leichty and Brisson 2014), and protocols for sWGA have been successfully developed to amplify and sequence malaria parasite genomes from contaminating host tissues (Guggisberg et al 2016; Oyola et al 2016; Sundararaman et al 2016; Cowell et al 2017).…”

Section: Introductionmentioning

confidence: 99%

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Genetic mapping of fitness determinants across the malaria parasitePlasmodium falciparumlife cycle

Kumar

McDew-White

et al. 2019

Preprint

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28Malaria is transmitted through female Anopheline mosquitoes where gamete fusion and meiosis 29 occurs, and humans where parasites proliferate asexually. We describe a powerful approach to 30 identify the genetic determinants of parasite fitness across both invertebrate and vertebrate life-31 cycle stages in human malaria parasite Plasmodium falciparum using bulk segregant analysis 32 (BSA). We combined experimental genetic crosses using humanized mice, with selective whole 33 genome amplification and BSA at multiple developmental stages in both mosquito and vertebrate 34 host to examine parasite competition and identify genomic regions under selection. We generated 35 crosses between artemisinin resistant (ART-R, kelch13-C580Y) and ART-sensitive (ART-S, 36 kelch13-WT) parasite clones recently isolated from Southeast Asian patients. We then quantified 37 genome-wide changes in allele frequency in the parasite progeny population from infected midgut 38 and salivary glands of Anopheles stephensi mosquitoes, infected livers, emerging merozoites and 39 aliquots of in vitro cultured progeny parasites at intervals over 30 days. Three striking results 40 emerge: we observed (i) a strong skew (>80%) towards alleles from the ART-R parent in the 41 mosquito stage, that dropped to ~50% in the blood stage as selfed ART-R parasites were selected 42 against; (ii) highly repeatable skews in allele frequencies across the genome in blood stage 43 parasites; (iii) particularly strong selection (selection coefficient (s) ≤ 0.18/asexual cycle) against 44 alleles from the ART-R parent at loci on chromosome 12 containing MRP2 and chromosome 14 45 containing ARPS10. This approach robustly identifies selected loci and has strong potential for 46 identifying parasite genes that interact with the mosquito vector or compensatory loci involved in 47 drug resistance. 48 49 3 50 Parasitic organisms frequently use multiple hosts and have several morphologically and 51 transcriptionally distinctive life cycle stages. Within each host, parasites must circumvent immune 52 defenses and navigate to new tissues. There are frequently extreme bottlenecks in parasite numbers 53 during transmission (Hopp et al. 2015), with rapid proliferative growth within hosts, and intense 54 competition between co-infecting parasite genotypes. For example, the life cycle of malaria 55 parasites involves successive infection of two hosts: female Anopheles mosquitoes, where gamete 56 fusion, meiosis and recombination occurs, and humans in which parasites travel from the skin, 57 develop in the liver and then proliferate asexually in the blood stream. Ideally, we would like to 58 understand how natural selection operates across the complete life cycle and document the genes 59 subject to selection pressures at each life cycle stage: during erythrocytic growth, gametocyte 60 production, oocyst development in the mosquito midgut, migration of sporozoites to the salivary 61 glands, transmission from the salivary glands, sporozoite survival in the skin, and est...

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Section: Discussionsupporting

confidence: 54%

Section: Introductionmentioning

confidence: 99%

Genetic mapping of fitness determinants across the malaria parasitePlasmodium falciparumlife cycle

Kumar

McDew-White

et al. 2019

Preprint

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“…The next druggable targets in the apicoplast were several prokaryotic metabolic pathways. Fosmidomycin, an inhibitor of MEP isoprenoid precursor biosynthesis in the apicoplast, causes parasite growth inhibition in a single replication cycle in vitro and rapid parasite clearance in human clinical trials ( Jomaa et al, 1999 ; Oyakhirome et al, 2007 ; Lanaspa et al, 2012 ; Guggisberg et al, 2016 ). Unfortunately initial parasite clearance is followed by recrudescent infections in 50% of patients.…”

Section: Introductionmentioning

confidence: 99%

Small molecule inhibition of apicomplexan FtsH1 disrupts plastid biogenesis in human pathogens

Amberg-Johnson

Hari

Ganesan

et al. 2017

eLife

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The malaria parasite Plasmodium falciparum and related apicomplexan pathogens contain an essential plastid organelle, the apicoplast, which is a key anti-parasitic target. Derived from secondary endosymbiosis, the apicoplast depends on novel, but largely cryptic, mechanisms for protein/lipid import and organelle inheritance during parasite replication. These critical biogenesis pathways present untapped opportunities to discover new parasite-specific drug targets. We used an innovative screen to identify actinonin as having a novel mechanism-of-action inhibiting apicoplast biogenesis. Resistant mutation, chemical-genetic interaction, and biochemical inhibition demonstrate that the unexpected target of actinonin in P. falciparum and Toxoplasma gondii is FtsH1, a homolog of a bacterial membrane AAA+ metalloprotease. PfFtsH1 is the first novel factor required for apicoplast biogenesis identified in a phenotypic screen. Our findings demonstrate that FtsH1 is a novel and, importantly, druggable antimalarial target. Development of FtsH1 inhibitors will have significant advantages with improved drug kinetics and multistage efficacy against multiple human parasites.

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“…To overcome the challenges of low sample quality and quantity, and to allow timely genetic analysis of clinical samples collected directly from patients without culture adaptation, we have used an approach that selectively amplifies parasite DNA from low blood volume clinical samples. The selective whole genome amplification (sWGA) strategy, originally described by Leichty and Brisson [12], uses computationally selected short oligonucleotide probes of 8--12 mers as primers that preferentially bind to the target genome, and this approach has been successfully applied to Laverania parasites including P. falciparum [13,14]. The purpose of the present study was to undertake a detailed evaluation of sWGA approaches for sequencing the P. falciparum genome from dried blood spots.…”

Section: Introductionmentioning

confidence: 99%

Whole genome sequencing ofPlasmodium falciparumfrom dried blood spots using selective whole genome amplification

Oyola

Ariani

Hamilton

et al. 2016

Preprint

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Background: Translating genomic technologies into healthcare applications for the malaria parasite Plasmodium falciparum has been limited by the technical and logistical difficulties of obtaining high quality clinical samples from the field. Sampling by dried blood spot (DBS) finger-pricks can be performed safely and efficiently with minimal resource and storage requirements compared with venous blood (VB). Here, the use of selective whole genome amplification (sWGA) to sequence the P. falciparum genome from clinical DBS samples was evaluated, and the results compared with current methods that use leucodepleted VB.Methods: Parasite DNA with high (>95%) human DNA contamination was selectively amplified by Phi29 polymerase using short oligonucleotide probes of 8-12 mers as primers. These primers were selected on the basis of their differential frequency of binding the desired (P. falciparum DNA) and contaminating (human) genomes.Results: Using sWGA method, clinical samples from 156 malaria patients, including 120 paired samples for head-tohead comparison of DBS and leucodepleted VB were sequenced. Greater than 18-fold enrichment of P. falciparum DNA was achieved from DBS extracts. The parasitaemia threshold to achieve >5× coverage for 50% of the genome was 0.03% (40 parasites per 200 white blood cells). Over 99% SNP concordance between VB and DBS samples was achieved after excluding missing calls. Conclusion:The sWGA methods described here provide a reliable and scalable way of generating P. falciparum genome sequence data from DBS samples. The current data indicate that it will be possible to get good quality sequence on most if not all drug resistance loci from the majority of symptomatic malaria patients. This technique overcomes a major limiting factor in P. falciparum genome sequencing from field samples, and paves the way for large-scale epidemiological applications.

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Whole-Genome Sequencing to Evaluate the Resistance Landscape Following Antimalarial Treatment Failure With Fosmidomycin-Clindamycin

Cited by 31 publications

References 45 publications

Genetic mapping of fitness determinants across the malaria parasitePlasmodium falciparumlife cycle

Genetic mapping of fitness determinants across the malaria parasitePlasmodium falciparumlife cycle

Small molecule inhibition of apicomplexan FtsH1 disrupts plastid biogenesis in human pathogens

Whole genome sequencing ofPlasmodium falciparumfrom dried blood spots using selective whole genome amplification

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