Biology subjects

Koepfli, C.

Publications and source records attributed to Koepfli, C..

8 recordsLinked to original sources

bioRxiv2023

Plasmodium falciparum transmission in the highlands of Ethiopia is driven by closely related and clonal parasites

Malaria cases are frequently recorded in the Ethiopian highlands even at altitudes above 2,000 m. The epidemiology of malaria in the Ethiopian highlands, and in particular the role of importation by human migration from the highly endemic lowlands is not well understood. We characterized the parasite population structure and genetic relatedness by sequencing 159 P. falciparum samples from Gondar and an additional 28 samples from Ziway using a highly multiplexed droplet digital PCR (ddPCR)-based amplicon deep sequencing method targeting 35 microhaplotypes and drug resistance loci. Diversity was moderate (mean HE: 0.54), and infection complexity was low (74.9% single clone infections). A significant percentage of infections shared genomic haplotypes, even across transmission seasons, indicating persistent local and focal transmission. Multiple clusters of clonal or near-clonal infections were identified, highlighting the overall high genetic relatedness. Frequently, infections from travelers were the earliest observed cases, suggesting that parasites may have been imported and then transmitted locally. We observed population structure between Gondar and Ziway, although some haplotypes were shared between sites. 31.1% of infections carried pfhrp2 deletions and 84.4% pfhrp3 deletions, and 28.7% pfhrp2/pfhrp3 double deletions. Parasites with pfhrp2/3 deletions and wild-type parasites were genetically distinct. Mutations associated with resistance to sulfadoxine-pyrimethamine and lumefantrine were observed at near-fixation, but no mutations in pfk13 were found. In conclusion, genomic data corroborates local transmission and the importance of intensified control in the Ethiopian highlands.

genomics↗

bioRxiv2023

Highly multiplexed ddPCR-amplicon sequencing reveals strong Plasmodium falciparum population structure and isolated populations amenable to local elimination efforts in Zanzibar

Over the past 15 years, Zanzibar has made great strides towards malaria elimination; yet progress has stalled. Parasite genetic data of Plasmodium falciparum may inform strategies for malaria elimination by helping to identify contributory factors to parasite persistence. Here we elucidate fine-scale parasite population structure and infer relatedness and connectivity of infections using an identity-by-descent (IBD) approach. We sequenced 518 P. falciparum samples from 5 districts covering both main islands using a novel, highly multiplexed droplet digital PCR (ddPCR)-based amplicon deep sequencing method targeting 35 microhaplotypes and drug-resistance loci. Despite high genetic diversity, we observe strong fine-scale spatial and temporal structure of local parasite populations, including isolated populations on Pemba Island and genetically admixed populations on Unguja Island, providing evidence of ongoing local transmission. We observe a high proportion of highly related parasites in individuals living closer together, including between clinical index cases and the mostly asymptomatic cases surrounding them, consistent with isolation-by-distance. We identify a substantial fraction (2.9%) of related parasite pairs between Zanzibar, and mainland Tanzania and Kenya, consistent with recent importation. We identify haplotypes known to confer resistance to known antimalarials in all districts, including multidrug-resistant parasites, but most parasites remain sensitive to current first-line treatments. Our study provides a high-resolution view of parasite genetic structure across the Zanzibar archipelago and reveals actionable patterns, including isolated parasite populations, which may be prioritized for malaria elimination.

genomics↗

bioRxiv2021

Systematic review of Plasmodium falciparum and Plasmodium vivax polyclonal infections: Impact of prevalence, study population characteristics, and laboratory procedures

Multiple infections of genetically distinct clones of the same Plasmodium species are common in many malaria endemic settings. Mean multiplicity of infection (MOI) and the proportion of polyclonal infections are often reported as surrogate marker of transmission intensity, yet the relationship with traditional measures such as parasite prevalence is not well understood. We have searched Pubmed for articles on P. falciparum and P. vivax multiplicity, and compared the proportion of polyclonal infections and mean MOI to population prevalence. The impact of the genotyping method, number of genotyping markers, method for diagnosis (microscopy/RDT vs. PCR), presence of clinical symptoms, age, geographic region, and year of sample collection on multiplicity indices were assessed. For P. falciparum, 153 studies met inclusion criteria, yielding 275 individual data points and 33526 genotyped individuals. The proportion of polyclonal infections ranged from 0-96%, and mean MOI from 1-6.1. For P. vivax, 54 studies met inclusion criteria, yielding 115 data points and 13325 genotyped individuals. The proportion of polyclonal infections ranged from 0-100%, and mean MOI from 1-3.8. For both species, the proportion of polyclonal infections ranged from very low to close to 100% at low prevalence, while at high prevalence it was always high. Each percentage point increase in prevalence resulted in a 0.34% increase in the proportion of polyclonal P. falciparum infections (P<0.001), and a 0.78% increase in the proportion of polyclonal P. vivax infections (P<0.001). In multivariable analysis, higher prevalence, typing multiple markers, diagnosis of infections by PCR, and sampling in Africa were found to result in a higher proportion of P. falciparum polyclonal infections. For P. vivax, prevalence, year of study, typing multiple markers, and geographic region were significant predictors. In conclusion, polyclonal infections are frequently present in all settings, but the association between multiplicity and prevalence is weak.

microbiology↗

bioRxiv2020

Plasmodium falciparum increases its investment in gametocytes in the wet season in asymptomatic individuals

In many regions, malaria transmission is seasonal, but it is not well understood whether P. falciparum modulates its investment in transmission in response to seasonal vector abundance. In two sites in western Kenya (Chulaimbo and Homa Bay), we sampled 1116 asymptomatic individuals in the wet season, when vectors are abundant, and 1743 in the dry season. We screened for P. falciparum by qPCR, and gametocytes by pfs25 RT-qPCR. Parasite prevalence in Chulaimbo and Homa Bay was 27.1% and 9.4% in the dry season, and 48.2% and 7.8% in the wet season respectively. Mean parasite densities did not differ between seasons (P=0.562). A contrasting pattern of gametocyte carriage was observed. In the wet season, fewer infections harbored gametocytes (22.3% vs. 33.8%, P=0.009), but densities were 3-fold higher (P<0.001). Thus, in the wet season, among gametocyte positive individuals, higher proportion of all parasites were gametocytes, reflecting an increased investment in transmission.

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

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↗