Biology subjects

Koepfli, C.

Publications and source records attributed to Koepfli, C..

4 recordsLinked to original sources

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

Integrated transcriptomic, proteomic and epigenomic analysis of Plasmodium vivax salivary-gland sporozoites

Plasmodium vivax is the key obstacle to malaria elimination in Asia and Latin America, largely attributed to its ability to form resilient hypnozoites (sleeper-cells) in the host liver that escape treatment and cause relapsing infections. The decision to form hypnozoites is made early in the liver infection and may already be set in sporozoites prior to invasion. To better understand these early stages of infection, we undertook a comprehensive transcriptomic and histone epigenetic characterization of P. vivax sporozoites. The salivary-gland sporozoite transcriptome is heavily composed of transcripts associated with functions needed for early infection of the vertebrate host and development within hepatocytes. Through comparisons to recently published proteome data for the P. vivax sporozoite, our study finds that although highly transcribed, these transcripts are not detectable as proteins and may be regulated through translational repression; a finding we test for a small subset of transcripts and proteins through immunofluorescent microscopy of sporozoites and liver stages in humanized mice. We identify differential transcription between the sporozoite and published transcriptomes of asexual blood-stages and mixed versus hypnozoite-enriched liver stages. These comparisons point to multiple layers of transcriptional, post-transcriptional and post-translational control that appear active in sporozoites and to a lesser extent hypnozoites, but largely absent in replicating liver schizonts or mixed blood-stages. Common transcripts up-regulated in sporozoites and hypnozoites compared to mixed (i.e., schizont) liver-stages identify genes linked to dormancy/persistence in bacteria, amoebae and plants. We also characterise histone epigenetic modifications in the P. vivax sporozoite and explore their role in regulating transcription. Collectively, these data support the hypothesis that the sporozoite as a tightly programmed stage primed to infect the human host and identifies potential mechanisms for hypnozoite-formation that may be further explored in liver stage models.

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↗

bioRxiv2017

Long-term sustained malaria control leads to inbreeding and fragmentation of Plasmodium vivax populations

The human malaria parasite Plasmodium vivax is resistant to malaria control strategies maintaining high genetic diversity even when transmission is low. To investigate whether declining P. vivax transmission leads to increasing P. vivax population structure that would facilitate elimination, we genotyped samples from a wide range of transmission intensities and spatial scales in the Southwest Pacific, including two time points at one site (Tetere, Solomon Islands) during intensified control. Analysis of 887 P. vivax microsatellite haplotypes from hyperendemic Papua New Guinea (PNG, n = 443), meso-hyperendemic Solomon Islands (n= 420), and hypoendemic Vanuatu (n=24) revealed increasing population structure and multilocus linkage disequilibrium and a modest decline in diversity as transmission decreases over space and time. In Solomon Islands, which has had sustained control efforts for 20 years, and Vanuatu, which has experienced sustained low transmission for many years, significant population structure was observed at different spatial scales. We conclude that control efforts will eventually impact P. vivax population structure and with sustained pressure, populations may eventually fragment into a limited number of clustered foci that could be targeted for elimination.

epidemiology↗