Molecular approaches to determine the multiplicity of Plasmodium infections

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.

Diversity and Multiplicity of P. falciparum infections among asymptomatic school children in Mbita, Western Kenya

“…However, the high prevalence of asymptomatic carriers especially among older children 19,[24][25][26][27][28] , high recombination rates among distinct P. falciparum clones in endemic settings leading to emergence of highly diverse parasite isolates, rapid emergence and distribution of drug resistant P. falciparum parasite strains [29][30][31][32][33] , and prevalence of infections characterized by multiple genetically distinct parasite strains are some of the major contributing factors hindering the global malaria control and elimination 31,34,35 . In highly endemic environments, many individuals carry multiple parasite clones [36][37][38] . This may have both positive and negative implications in the fight against malaria.…”

“…These indices of P. falciparum parasite infections are also used in determining the impact of some key malaria interventions like drug and vaccine efficacy studies 29,47,48 . Numerous studies about multiplicity and diversity of P. falciparum infections have been carried out in various WHO regions using different tools including size-polymorphic antigens and microsatellite markers followed by agarose gel and capillary electrophoresis respectively, single nucleotide polymorphism genotyping, amplicon ultra-deep sequencing and whole genome sequencing 36,49 . The use of molecular genotyping of polymorphic antigenic markers like merozoite surface protein (MSP) 1 and 2, glutamate rich protein (Glurp) for parasite diversity studies are faced with some criticisms due to the fact that these genes are implicated to be under strong immune selection 44,[50][51][52] .…”

“…Microsatellite markers are highly polymorphic, widely distributed in the Plasmodium genome, perceived to be selection neutral and readily amplifiable using cheap methods like PCR 35,53,54 . These criteria have made them an ideal tool for the estimation of parasite genetic diversity and MOI 36,46,48 .…”

Detection of low-density Plasmodium falciparum infections using amplicon deep sequencing

“…Within each sample, haplotypes were filtered according to a set of pre-specified thresholds developed by Neafsey et al [1]. Haplotypes were required to (1) cover the entire amplicon region, (2) have no uncalled bases, (3) be supported by at least two sets of merged read pairs (henceforth referred to simply as "reads"), and (4) have an intra-sample frequency ≥ 0.01. To account for potential PCR and sequencing errors, the filtered haplotypes were clustered based on nucleotide distance and read depth.…”

“…When targeting a highly polymorphic genomic region, a single amplicon can distinguish among hundreds of unique haplotypes [1], providing higher resolution than either SNP-based or length-based genotyping approaches. This improves estimates of the number of lineages within polyclonal infections (or complexity of infection; COI) [2][3][4], permits the discovery of unknown alleles [5][6][7], and provides increased information for haplotype-based analyses of epistasis and linkage disequilibrium [8].…”