LSTM Home > LSTM Research > LSTM Online Archive

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, Rebecca M, Zhou, Zhiyong, Sheth, Mili, Sergent, Sheila, Frace, Michael, Nayak, Vishal, Hu, Bin, Gimnig, John, terKuile, Feiko ORCID:, Lindblade, Kim, Slutsker, Laurence, Hamel, Mary J, Desai, Meghna, Otieno, Kephas, Kariuki, Simon, Vigfusson, Ymir and Shi, Ya Ping (2021) '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.'. Malaria Journal, Vol 20, p. 92.

barcode PCR A Roberts.pdf - Published Version
Available under License Creative Commons Attribution.

Download (1MB) | Preview


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.

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.

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.

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.

Item Type: Article
Subjects: QU Biochemistry > Genetics > QU 450 General Works
QV Pharmacology > Drug Standardization. Pharmacognosy. Medicinal Plants > QV 771 Standardization and evaluation of drugs
QX Parasitology > Protozoa > QX 135 Plasmodia
QX Parasitology > QX 4 General works
WC Communicable Diseases > Tropical and Parasitic Diseases > WC 750 Malaria
Faculty: Department: Clinical Sciences & International Health > Clinical Sciences Department
Digital Object Identifer (DOI):
Depositing User: Tracy Seddon
Date Deposited: 25 Mar 2021 14:40
Last Modified: 25 Mar 2021 14:40


View details

Actions (login required)

Edit Item Edit Item