Using Amplicon Deep Sequencing to Detect Genetic Signatures ofPlasmodium vivaxRelapse

Abstract

Plasmodium vivax infections often recur due to relapse of hypnozoites from the liver. In malaria-endemic areas, tools to distinguish relapse from reinfection are needed. We applied amplicon deep sequencing to P. vivax isolates from 78 Cambodian volunteers, nearly one-third of whom suffered recurrence at a median of 68 days. Deep sequencing at a highly variable region of the P. vivax merozoite surface protein 1 gene revealed impressive diversity-generating 67 unique haplotypes and detecting on average 3.6 cocirculating parasite clones within individuals, compared to 2.1 clones detected by a combination of 3 microsatellite markers. This diversity enabled a scheme to classify over half of recurrences as probable relapses based on the low probability of reinfection by multiple recurring variants. In areas of high P. vivax diversity, targeted deep sequencing can help detect genetic signatures of relapse, key to evaluating antivivax interventions and achieving a better understanding of relapse-reinfection epidemiology.

Pooled Amplicon Deep Sequencing of Candidate Plasmodium falciparum Transmission-Blocking Vaccine Antigens

“…Library preparation, sequencing, and analysis of Pfs25 haplotypes was done as previously described (Supplemental Material). 16 Part of the pfs48/45 gene (nucleotides 706-1301 [amino acids 236-433] of PF3D7_1346700) was amplified using non-barcoded primers, and each pool was indexed and library prepared individually as previously described in a study. 11 The reaction consisted of 10X Roche FastStart Hi-Fidelity Buffer, 400 nM forward primer (CAAGAAGGAAAAGAAAAAG CCTTA), 400 nM reverse primer (GCCAAAAATCCATAAT ATGCTGA), 10 nM dNTPs, 0.5 μL Roche FastStart Hi-Fidelity Enzyme, and 5 μL DNA in a 50-μL volume.…”

The utility of genomic data forPlasmodium vivaxpopulation surveillance

“…Amplicon sequencing approaches can also be used to interpret individual parasite types among polygenomic infections. 23 Overall, genetic markers help characterise individual populations and can reveal genetic relatedness between populations. The combined knowledge of population structure and parasite relatedness within geographical areas allows identification of parasite lineages and tracking of their movement within and beyond these regions.…”

“…Relapse plays an important role in P. vivax transmission dynamics, 24 in initiating the transmission season in seasonal transmission settings (such as Southern Mexico 51 ), contributes to high levels of genetic diversity in populations, and is responsible for a high percentage of P. vivax cases, for example more than 50% in a cohort in Cambodia. 23 It also confounds monitoring for treatment failures due to resistance, as the reappearance of blood stage infection can also be due to the emergence of previously latent parasites. 25 Relapse parameters, such as the proportion of infections that result in relapse, the period of time to first relapse, and the time between subsequent relapses, are therefore critical to understanding P. vivax epidemiology.…”

“…Three principal explanations for the high proportion of relapses that have complex infections containing heterologous or novel genotypes have been investigated with genomic or genotyping data. 23 The first possible explanation is that a long history of P. vivax exposure in endemic areas causes a reservoir of diverse hypnozoites to accumulate in the liver, from which a variable subset of clones activates upon relapse. The genotypes present in the relapsed infection therefore can be diverse and dissimilar from the primary infection observed when study began.…”

“…The second explanation, minority variant expansion, has been observed when using high-throughput DNA sequencing techniques to attain high sequencing coverage of a polymorphic amplicon. 23 Here, variants in a complex infection that are below the detection threshold of less sensitive methods such as microsatellite typing go unobserved in the primary infection, but are more abundant or predominant in later recurrences. Finally, recombination in the mosquito midgut prior to inoculation can result in an infection comprised of differing, but closely related relatives.…”