Amplicon deep sequencing improves Plasmodium falciparum genotyping in clinical trials of antimalarial drugs

Gruenberg, Maria; Lerch, Anita; Beck, Hans‐Peter; Felger, Ingrid

doi:10.1038/s41598-019-54203-0

Cited by 49 publications

(67 citation statements)

References 30 publications

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“…Nevertheless, the results from this study showed a substantial improvement in sensitivity for detecting minor parasite populations in a sample, particularly those above 5% proportion, with acceptable precision. While deep sequencing of individual targeted antigenic genes also provides ability to detect gene-specific minor variants in a sample, the estimates in the highly selected genes might not represent true genomic signatures of parasites [ 14 , 15 , 37 ] and may offer limited temporal and geographic discrimination between parasite populations [ 15 ].…”

Section: Discussionmentioning

confidence: 99%

Development of a new barcode-based, multiplex-PCR, next-generation-sequencing assay and data processing and analytical pipeline for multiplicity of infection detection of Plasmodium falciparum

Mitchell

Zhou

Sheth³

et al. 2021

Malar J

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Background Simultaneous infection with multiple malaria parasite strains is common in high transmission areas. Quantifying the number of strains per host, or the multiplicity of infection (MOI), provides additional parasite indices for assessing transmission levels but it is challenging to measure accurately with current tools. This paper presents new laboratory and analytical methods for estimating the MOI of Plasmodium falciparum. Methods Based on 24 single nucleotide polymorphisms (SNPs) previously identified as stable, unlinked targets across 12 of the 14 chromosomes within P. falciparum genome, three multiplex PCRs of short target regions and subsequent next generation sequencing (NGS) of the amplicons were developed. A bioinformatics pipeline including B4Screening pathway removed spurious amplicons to ensure consistent frequency calls at each SNP location, compiled amplicons by SNP site diversity, and performed algorithmic haplotype and strain reconstruction. The pipeline was validated by 108 samples generated from cultured-laboratory strain mixtures in different proportions and concentrations, with and without pre-amplification, and using whole blood and dried blood spots (DBS). The pipeline was applied to 273 smear-positive samples from surveys conducted in western Kenya, then providing results into StrainRecon Thresholding for Infection Multiplicity (STIM), a novel MOI estimator. Results The 24 barcode SNPs were successfully identified uniformly across the 12 chromosomes of P. falciparum in a sample using the pipeline. Pre-amplification and parasite concentration, while non-linearly associated with SNP read depth, did not influence the SNP frequency calls. Based on consistent SNP frequency calls at targeted locations, the algorithmic strain reconstruction for each laboratory-mixed sample had 98.5% accuracy in dominant strains. STIM detected up to 5 strains in field samples from western Kenya and showed declining MOI over time (q < 0.02), from 4.32 strains per infected person in 1996 to 4.01, 3.56 and 3.35 in 2001, 2007 and 2012, and a reduction in the proportion of samples with 5 strains from 57% in 1996 to 18% in 2012. Conclusion The combined approach of new multiplex PCRs and NGS, the unique bioinformatics pipeline and STIM could identify 24 barcode SNPs of P. falciparum correctly and consistently. The methodology could be applied to field samples to reliably measure temporal changes in MOI.

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Section: Discussionmentioning

confidence: 99%

Development of a new barcode-based, multiplex-PCR, next-generation-sequencing assay and data processing and analytical pipeline for multiplicity of infection detection of Plasmodium falciparum

Mitchell

Zhou

Sheth³

et al. 2021

Malar J

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“…A common genotyping method is to PCR amplify microsatellite markers or polymorphic genes, such as the msp family, the size of the resulting amplicon(s) being variable between different isolates [ 1 ]. Amplicon ultra-deep sequencing now allows multiplexing samples and are particularly useful to determine the proportion of each strain in infections with multiple genotypes [ 2 ]. Alternatively, genotyping a few dozen single nucleotide polymorphisms (SNPs) generates a molecular barcode specific to each isolate.…”

Section: Genotyping Methodsmentioning

confidence: 99%

Order within chaos: Harnessing Plasmodium falciparum var gene extreme polymorphism for malaria epidemiology

Guery

Claessens

2021

PLoS Genet

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“…The copyright holder for this preprint this version posted March 23, 2021. ; https://doi.org/10.1101/2021.03.23.436602 doi: bioRxiv preprint allelic diversity obtained from (15). We use the first three markers by default but also included the less diverse markers csp and msp-7 in our simulations as these may be used when other markers fail to amplify (15) and allows us to evaluate whether increasing the number of Ampseq markers would significantly improve accuracy.…”

Section: Genetic Diversity Of Infections At Treatment Is Termed the Mmentioning

confidence: 99%

“…AmpSeq methods are well established in the wider malarial context for tracking specific genes (e.g., for drug resistance) within populations (11)(12)(13), and for evaluating the efficacy of the RTS,S/AS01 vaccine (14). AmpSeq as a method to genotype malaria parasites in clinical trials is relatively novel (15) and, despite its putative advantages, has not yet been used as the primary genotyping endpoint for a clinical trial or TES (although it has been used to reanalyze archived blood samples (15)).…”

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confidence: 99%

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

Jones¹,

Kay

Hodel

et al. 2021

Preprint

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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.

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Amplicon deep sequencing improves Plasmodium falciparum genotyping in clinical trials of antimalarial drugs

Cited by 49 publications

References 30 publications

Development of a new barcode-based, multiplex-PCR, next-generation-sequencing assay and data processing and analytical pipeline for multiplicity of infection detection of Plasmodium falciparum

Development of a new barcode-based, multiplex-PCR, next-generation-sequencing assay and data processing and analytical pipeline for multiplicity of infection detection of Plasmodium falciparum

Order within chaos: Harnessing Plasmodium falciparum var gene extreme polymorphism for malaria epidemiology

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

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