Molecular approaches to determine the multiplicity of  Plasmodium  infections

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Abstract

Multiplicity of infection (MOI), also termed complexity of infection (COI), is defined as the number of genetically distinct parasite strains co-infecting a single host, which is an important indicator of malaria epidemiology. PCR-based genotyping often underestimates MOI. Next generation sequencing technologies provide much more accurate and genome-wide characterization of polyclonal infections. However, complete haplotype characterization of multiclonal infections remains a challenge due to PCR artifacts and sequencing errors, and requires efficient computational tools. In this review, the advantages and limitations of current molecular approaches to determine multiplicity of malaria parasite infection are discussed.

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Most cited references 108

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Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing

Magnus Manske, Olivo Miotto, Susana Campino … (2012)

Malaria elimination strategies require surveillance of the parasite population for genetic changes that demand a public health response, such as new forms of drug resistance. 1,2 Here we describe methods for large-scale analysis of genetic variation in Plasmodium falciparum by deep sequencing of parasite DNA obtained from the blood of patients with malaria, either directly or after short term culture. Analysis of 86,158 exonic SNPs that passed genotyping quality control in 227 samples from Africa, Asia and Oceania provides genome-wide estimates of allele frequency distribution, population structure and linkage disequilibrium. By comparing the genetic diversity of individual infections with that of the local parasite population, we derive a metric of within-host diversity that is related to the level of inbreeding in the population. An open-access web application has been established for exploration of regional differences in allele frequency and of highly differentiated loci in the P. falciparum genome.

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Standardizing methods to address clonality in population studies.

S Arnaud-Haond, C. M. Duarte, F Alberto … (2007)

Although clonal species are dominant in many habitats, from unicellular organisms to plants and animals, ecological and particularly evolutionary studies on clonal species have been strongly limited by the difficulty in assessing the number, size and longevity of genetic individuals within a population. The development of molecular markers has allowed progress in this area, and although allozymes remain of limited use due to their typically low level of polymorphism, more polymorphic markers have been discovered during the last decades, supplying powerful tools to overcome the problem of clonality assessment. However, population genetics studies on clonal organisms lack a standardized framework to assess clonality, and to adapt conventional data analyses to account for the potential bias due to the possible replication of the same individuals in the sampling. Moreover, existing studies used a variety of indices to describe clonal diversity and structure such that comparison among studies is difficult at best. We emphasize the need for standardizing studies on clonal organisms, and particularly on clonal plants, in order to clarify the way clonality is taken into account in sampling designs and data analysis, and to allow further comparison of results reported in distinct studies. In order to provide a first step towards a standardized framework to address clonality in population studies, we review, on the basis of a thorough revision of the literature on population structure of clonal plants and of a complementary revision on other clonal organisms, the indices and statistics used so far to estimate genotypic or clonal diversity and to describe clonal structure in plants. We examine their advantages and weaknesses as well as various conceptual issues associated with statistical analyses of population genetics data on clonal organisms. We do so by testing them on results from simulations, as well as on two empirical data sets of microsatellites of the seagrasses Posidonia oceanica and Cymodocea nodosa. Finally, we also propose a selection of new indices and methods to estimate clonal diversity and describe clonal structure in a way that should facilitate comparison between future studies on clonal plants, most of which may be of interest for clonal organisms in general.

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Twelve microsatellite markers for characterization of Plasmodium falciparum from finger-prick blood samples.

T. J. Anderson, X Z Su, M. Bockarie … (1999)

Multiple, selectively neutral genetic markers are the most appropriate tools for analysis of parasite population structure and epidemiology, but yet existing methods for characterization of malaria field samples utilize a limited number of antigen encoding genes, which appear to be under strong selection. We describe protocols for characterization of 12 microsatellite markers from finger-prick blood samples infected with Plasmodium falciparum. A two-step, heminested strategy was used to amplify all loci, and products were visualized by fluorescent end-labelling of internal primers. This procedure allows amplification from low levels of template, while eliminating the problem of spurious products due to primer carry over from the primary round of PCR. The loci can be conveniently multiplexed, while accurate sizing and quantification of PCR products can be automated using the GENOTYPER software. The primers do not amplify co-infecting malaria species such as P. vivax and P. malariae. To demonstrate the utility of these markers, we characterized 57 infected finger-prick blood samples from the village of Mebat in Papua New Guinea for all 12 loci, and all samples were genotyped a second time to measure reproducibility. Numbers of alleles per locus range from 4 to 10 in this population, while heterozygosities range from 0.21 to 0.87. Reproducibility (measured as concordance between predominant alleles detected in replicate samples) ranged from 92 to 98% for the 12 loci. The composition of PCR products from infections containing multiple malaria clones could also be defined using strict criteria and scored in a highly repeatable manner.

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Author and article information

Contributors

Daibin Zhong:

ORCID: http://orcid.org/0000-0002-2771-9598

dzhong@uci.edu

Guiyun Yan: guiyuny@uci.edu

Journal

Journal ID (nlm-ta): Malar J

Journal ID (iso-abbrev): Malar. J

Title: Malaria Journal

Publisher: BioMed Central (London )

ISSN (Electronic): 1475-2875

Publication date (Electronic): 23 April 2018

Publication date PMC-release: 23 April 2018

Publication date Collection: 2018

Volume: 17

Electronic Location Identifier: 172

Affiliations

[1 ]ISNI 0000 0001 0668 7243, GRID grid.266093.8, Program in Public Health, , University of California, ; Irvine, CA 92617 USA

[2 ]ISNI 0000 0001 2097 4281, GRID grid.29857.31, Department of Entomology, , Pennsylvania State University, ; University Park, PA 16802 USA

Author information

Daibin Zhong http://orcid.org/0000-0002-2771-9598

Article

Publisher ID: 2322

DOI: 10.1186/s12936-018-2322-5

PMC ID: 5914063

PubMed ID: 29685152

SO-VID: bf29ed31-d0a7-44b9-b812-f55a3d8bd546

License:

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

History

Date received : 19 December 2017

Date accepted : 18 April 2018

Funding

Funded by: FundRef http://dx.doi.org/10.13039/100000002, National Institutes of Health;

Award ID: R01 AI050243

Award ID: D43 TW001505

Award ID: U19 AI129326

Award ID: U19 AI089672

Award Recipient : Liwang Cui Guiyun Yan

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ScienceOpen disciplines: Infectious disease & Microbiology

Keywords: malaria parasite,multiplicity of infection,molecular marker,next generation sequencing,amplicon ultra-deep sequencing,single nucleotide polymorphism

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ScienceOpen disciplines: Infectious disease & Microbiology

Keywords: malaria parasite, multiplicity of infection, molecular marker, next generation sequencing, amplicon ultra-deep sequencing, single nucleotide polymorphism

Molecular approaches to determine the multiplicity of Plasmodium infections

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Abstract

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Most cited references 108

Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing

Standardizing methods to address clonality in population studies.

Twelve microsatellite markers for characterization of Plasmodium falciparum from finger-prick blood samples.

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