Biology subjects

Felger, I.

Publications and source records attributed to Felger, I..

5 recordsLinked to original sources

bioRxiv2021

Should deep-sequenced amplicons become the new gold-standard for analysing malaria drug clinical trials?

BackgroundRegulatory clinical trials are required to ensure the continued supply and deployment of effective antimalarial drugs. Patient follow-up in such trials typically lasts several weeks as the drugs have long half-lives and new infections often occur during this period. "Molecular correction" is therefore used to distinguish drug failures from new infections. The current WHO-recommend method for molecular correction uses length-polymorphic alleles at highly diverse loci but is inherently poor at detecting low density clones in polyclonal infections. This likely leads to substantial underestimates of failure rates, delaying the replacement of failing drugs with potentially lethal consequences. Deep sequenced amplicons (AmpSeq) substantially increase the detectability of low-density clones and may offer a new "gold standard" for molecular correction. MethodsPharmacological simulation of clinical trials was used to evaluate the suitability of AmpSeq for molecular correction. We investigated the impact of factors such as the number of amplicon loci analysed, the informatics criteria used to distinguish genotyping noise from real low density signals, the local epidemiology of malaria transmission, and the potential impact of genetic signals from gametocytes. ResultsAmpSeq greatly improved molecular correction and provided accurate drug failure rate estimates. The use of 3 to 5 amplicons was sufficient, and simple, non-statistical, criteria could be used to classify recurrent infections as drug failures or new infections. ConclusionsThese results strongly endorse the deployment of AmpSeq as the standard for molecular correction in regulatory trials, with its potential extension into routine surveillance once the requisite technical support becomes established.

microbiology↗

bioRxiv2019

Monitoring Plasmodium falciparum and Plasmodium vivax using microsatellite markers indicates limited changes in population structure after substantial transmission decline in Papua New Guinea

Monitoring the genetic structure of malaria parasite populations has been proposed as a novel and sensitive approach to quantify the impact of malaria control and elimination efforts. Here we describe the first population genetic analysis of sympatric Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) populations following nationwide distribution of long-lasting insecticide treated nets (LLIN) in Papua New Guinea (PNG). Parasite isolates from serial cross-sectional studies pre-(2005-6) and post-LLIN (2010-2014) were genotyped using microsatellite markers. Despite parasite prevalence declining substantially in these communities (East Sepik: Pf=54.9-8.5%, Pv=35.7-5.6%, Madang: Pf=38.0-9.0%, Pv: 31.8-19.7%), genetically diverse and intermixing parasite populations remained. P. falciparum diversity declined modestly post-LLIN relative to pre-LLIN (East Sepik: Rs = 7.1-6.4, He = 0.77-0.71; Madang: Rs= 8.2-6.1, He = 0.79-0.71). Unexpectedly, population structure present in pre-LLIN populations was lost post-LLIN, suggesting that more frequent human movement between provinces may have contributed to higher gene flow between provinces. P. vivax prevalence initially declined but increased again in one province, yet diversity remained high throughout the study period (East Sepik: Rs=11.4-9.3, He=0.83-0.80; Madang: Rs=12.2-14.5, He=0.85-0.88). Although genetic differentiation values increased between provinces over time, no significant population structure was observed at any time point. For both species, the emergence of clonal transmission and significant multilocus linkage disequilibrium (mLD) due to increased focal inbreeding post-LLIN was a strong indicator of impact on the parasite population using these markers. After eight years of intensive malaria control in PNG and substantial prevalence decline the impact on parasite population diversity and structure detectable by microsatellite genotyping was limited.

microbiology↗

bioRxiv2018

Longitudinal tracking of Plasmodium falciparum clones in complex infections by amplicon deep sequencing

BackgroundLongitudinal tracking of individual Plasmodium falciparum strains in multi-clonal infections is essential for investigating infection dynamics of malaria. The traditional genotyping techniques did not permit tracking changes in individual clone density during persistent natural infections. Amplicon deep sequencing (Amp-Seq) offers a tool to address this knowledge gap.\n\nMethodsThe sensitivity of Amp-Seq for relative quantification of clones was investigated using three molecular markers, ama1-D2, ama1-D3, and cpmp. Amp-Seq and length-polymorphism based genotyping were compared for their performance in following minority clones in longitudinal samples from Papua New Guinea.\n\nResultsAmp-Seq markers were superior to length-polymorphic marker msp2 in detecting minority clones (sensitivity Amp-Seq: 95%, msp2: 85%). Multiplicity of infection (MOI) by Amp-Seq was 2.32 versus 1.73 for msp2. The higher sensitivity had no effect on estimates of force of infection because missed minority clones were detected in preceding or succeeding bleeds. Individual clone densities were tracked longitudinally by Amp-Seq despite MOI>1, thus providing an additional parameter for investigating malaria infection dynamics.\n\nConclusionAmp-Seq based genotyping of longitudinal samples improves detection of minority clones and estimates of MOI. Amp-Seq permits tracking of clone density over time to study clone competition or the dynamics of specific, i.e. resistance-associated genotypes.

microbiology↗

bioRxiv2017

Human Migration And The Spread Of Malaria Parasites To The New World

BackgroundThe Americas were the last continent to be settled by modern humans, but how and when human malaria parasites arrived in the New World is uncertain. Here, we apply phylogenetic analysis and coalescent-based gene flow modeling to a global collection of Plasmodium falciparum and P. vivax mitogenomes to infer the demographic history and geographic origins of malaria parasites circulating in the Americas. Importantly, we examine P. vivax mitogenomes from previously unsampled forest-covered sites along the Atlantic Coast of Brazil, including the vivax-like species P. simium that locally infects platyrrhini monkeys.\n\nResultsThe best-supported gene flow models are consistent with migration of both malaria parasites from Africa and South Asia to the New World, with no genetic signature of a population bottleneck upon parasite's arrival in the Americas. We found evidence of additional gene flow from Melanesia in P. vivax (but not P. falciparum) mitogenomes from the Americas and speculate that some P. vivax lineages might have arrived with the Australasian peoples who contributed genes to Native Americans in pre-Columbian times. Mitochondrial haplotypes characterized in P. simium from monkeys from the Atlantic Forest are shared by local humans. These vivax-like lineages have not spread to the Amazon Basin, are much less diverse than P. vivax circulating elsewhere in Brazil, and show no close genetic relatedness with P. vivax populations from other continents.\n\nConclusionsEnslaved peoples brought from a wide variety of African locations were major carriers of P. falciparum mitochondrial lineages into the Americas, but additional human migration waves are likely to have contributed to the extensive genetic diversity of present-day New World populations of P. vivax. The reduced genetic diversity of vivax-like monkey parasites, compared with human P. vivax from across this country, argues for a recent human-to-monkey transfer of these lineages in the Atlantic Forest of Brazil.\n\nAuthor summaryMalaria is currently endemic to the Americas, with over 400,000 laboratory-confirmed infections reported annually, but how and when human malaria parasites entered this continent remains largely unknown. To determine the geographic origins of malaria parasites currently circulating in the Americas, we examined a global collection of Plasmodium falciparum and P. vivax mitochondrial genomes, including those from understudied isolates of P. vivax and P. simium, a vivax-like species that infect platyrrhini monkeys, from the Atlantic Forest of Brazil. We found evidence of significant historical migration to the New World of malaria parasites from Africa and, to a lesser extent, South Asia, with further genetic contribution of Melanesian lineages to South American P. vivax populations. Importantly, mitochondrial haplotypes of P. simium are shared by monkeys and humans from the Atlantic Forest, most likely as a result of a recent human-to-monkey transfer. Interestingly, these potentially zoonotic lineages are not found in the Amazon Basin, the main malaria-endemic area in the Americas. We conclude that enslaved Africans were the main carriers of P. falciparum mitochondrial lineages into the Americas, whereas additional migration waves of Australasian peoples and parasites may have contributed to the genetic makeup of present-day New World populations of P. vivax.

microbiology↗

bioRxiv2017

Development Of Amplicon Deep Sequencing Markers And Data Analysis Pipeline For Genotyping Multi-Clonal Malaria Infections

Amplicon deep sequencing permits sensitive detection of minority clones and improves discriminatory power for genotyping multi-clone Plasmodium falciparum infections. Such high resolution is needed for molecular monitoring of drug efficacy trials. Targeted sequencing of molecular marker csp and novel marker cpmp was conducted in duplicate on mixtures of parasite culture strains and 37 field samples. A protocol to multiplex up to 384 samples in a single sequencing run was applied. Software \"HaplotypR\" was developed for data analysis. Cpmp was highly diverse (He=0.96) in contrast to csp (He=0.57). Minority clones were robustly detected if their frequency was >1%. False haplotype calls owing to sequencing errors were observed below that threshold. To reliably detect haplotypes at very low frequencies, experiments are best performed in duplicate and should aim for coverage of >10000 reads/amplicon. When compared to length polymorphic marker msp2, highly multiplexed amplicon sequencing displayed greater sensitivity in detecting minority clones.

microbiology↗