Biology subjects

Lerch, A.

Publications and source records attributed to Lerch, A..

8 recordsLinked to original sources

bioRxiv2026

Estimation of adult census size from close-kin dyads in the malaria mosquito Anopheles gambiae

Accurate estimates of adult mosquito abundance are central to the design and evaluation of vector control strategies, yet they are difficult to obtain from natural populations. Conventional mark-recapture methods for estimating adult mosquito census size pose logistical and other challenges. A recently developed close-kin mark-recapture (CKMR) approach is a promising alternative. However, application of CKMR has been largely confined to long-lived vertebrates. Validation on empirical data from short-lived, highly fecund insects is lacking. Here, we apply CKMR to a natural population of Anopheles gambiae, the primary African malaria mosquito, sampled from a small island in Lake Victoria, Uganda. Using a high-diversity amplicon panel of genome-wide markers, we genotyped 714 adult mosquitoes collected over a 20-day period. We classified pairs to close-kin categories by implementing probabilistic latent-kinship estimation rather than using a deterministic threshold-based framework. We observe numerous full-sibling pairs but no parent-offspring pairs, a pattern indicative of extreme variance in reproductive success owing to frequent failure of mosquito egg clutches to produce adults. To adapt the CKMR framework to mosquito life history, we explicitly modeled the mosquito life cycle including the possibility of clutch failure. By incorporating probabilities of both parent-offspring and full-sibling relationships, we estimate an adult female census size of 26,887 (95% credible interval: 6,979 - 146,011), and a clutch failure probability of 97.6% (CrI: 91.3 - 99.6%). We use these estimates to calculate the predicted variance in reproductive success and effective population size. Through individual-based simulations we confirm our estimates and the necessity of modeling reproductive variance to explain observed kinship patterns. Our results demonstrate that CKMR can be applied successfully to mosquitoes, provided that appropriate adjustments are made to account for their natural history.

ecology↗

bioRxiv2024

Multiplexed nanopore amplicon sequencing to distinguish recrudescence from new infection in antimalarial drug trials

BackgroundThe assessment of antimalarial drug efficacy against Plasmodium falciparum requires PCR correction to distinguish recrudescence from new infections by comparing parasite genotypes before treatment and in recurrent infections. Nanopore sequencing offers a low-cost, portable, scalable, and rapid alternative to traditional methods, supporting the expansion and decentralization of sequencing in endemic, resource-limited settings, potentially providing rapid PCR-corrected drug failure estimates. MethodsWe optimized a multiplexed AmpSeq panel targeting six microhaplotypes for high and uniform coverage. We assessed sensitivity and specificity for detecting minority clones in polyclonal infections and evaluated genetic diversity across the microhaplotype markers. We used mixtures of four P. falciparum laboratory strains at different ratios and 20 paired patient samples from a clinical trial. A custom bioinformatics workflow was used to infer haplotypes from polyclonal infections, including minority clones, with defined cut-off criteria for accurate haplotype calling. FindingsThe nanopore AmpSeq assay achieved uniform and high read coverage across all six microhaplotype markers (median coverage: 12,989x to 15,440x for laboratory strain mixtures and 7,011x to 11,600x for patients samples, respectively). We found high sensitivity in detecting minority clones (up to 50:1:1:1 in the 3D7:K1:HB3:FCB1 laboratory strain mixtures) and high specificity with less than 0.01% of all reads being false-positive haplotypes. Genetic diversity in the markers used was high (HE [≥] 0.98 and up to 31 unique haplotypes in 20 paired samples with cpmp), and concordant results in classifying new infections and recrudescence across all markers used were observed in 18 (90%) of 20 paired samples. InterpretationOur study demonstrates the feasibility of nanopore AmpSeq for distinguishing recrudescence from new infections in clinical trials. FundingSwiss Tropical and Public Health Institute.

molecular biology↗

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

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↗

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↗