Understanding Plasmodium vivax recurrent infections using an amplicon deep sequencing assay, PvAmpSeq, identity-by-descent and model-based classification

Abstract

Plasmodium vivax infections are characterised by recurrent bouts of blood-stage parasitaemia. Understanding the genetic relatedness of recurrences can distinguish whether these are caused by relapse, reinfection, or recrudescence, which is critical to understand treatment efficacy and transmission dynamics. We developed PvAmpseq, an amplicon sequencing assay targeting 11 SNP-rich regions of the P. vivax genome. PvAmpSeq was validated on field isolates from a clinical trial in the Solomon Islands and a longitudinal observational cohort in Peru, and statistical models were applied for genetic classification of infection pairs. In the Solomon Islands trial, where participants received antimalarials at baseline, half of the recurrent infections were caused by parasites with >50% relatedness to the baseline infection, with statistical models classifying 25% and 25% as probable relapses and recrudescences, respectively. In the Peruvian cohort, 26% of recurrences were likely relapses. PvAmpSeq provides high-resolution genotyping to characterise P. vivax recurrences, offering insights into transmission and treatment outcomes.

Keywords: Plasmodium vivax; genetic diversity; microhaplotypes; recurrences; relapses.

Figure 1.

Marker coverage and sample parasite density. (A) Number of reads per marker on field samples. (B) Number of reads and parasite density of field samples. (C) Limit of detection for parasite density; in blue: markers that passed the quality control step; in grey: markers that failed the quality control step; empty bars: markers with no successful PCR amplification.

Figure 2.

Detection of minority clones in mock and artificial mixed infections. (A) The Y-axis shows the sample ratios detected in mock mixed infections. The X-axis shows the markers grouped by expected sample ratios in mock infections (1:1, 1:10, 1:50, 1:100, 1:500, 1:1000, 10:1, 50:1, 100:1, 500:1 and 1000:1). In blue: clones from sample AR079; in yellow: clones from sample AR093; in red or orange: contaminants clones. expec: expected results from mocked mixed infections. Markers sharing the same microhaplotypes between samples are not displayed. (B) The detectability of the minor clone under different numbers of reads and artificial mixture ratios. The X-axis represents the mixture dilutions (%), and the Y-axis represents the success rate (%) of detecting the minor clone. Coloured by amplicon marker.

Figure 3.

Genetic diversity of markers and multiplicity of infection of validation samples. (A) Markers were sorted by descending H_e. He: Expected heterozygosity calculated as the number of microhaplotypes per marker divided by microhaplotype frequency on Solomon Islands (n = 135) and Peruvian samples (n = 140). The number of microhaplotypes per marker is indicated on top of each bar. (B) Microhaplotype frequency per marker estimated on the Solomon Islands and Peruvian samples. Each colour indicates a different microhaplotype. (C) Estimated multiplicity of infection (MOI) in field samples as the highest number of distinct microhaplotypes by all markers. The Y-axis shows the within-host frequency of microhaplotypes per sample, calculated as the percentage of microhaplotype reads per sample. The X-axis shows the samples sorted by estimated MOI. Every sample is represented by a vertical bar. (D) Distribution of MOI in paired samples, where Day 0 represents the baseline infection (Solomon Islands, n = 41; Peru, n = 40) and Day X the day of recurrent infection (Solomon Islands, n = 58; Peru, n = 47).

Figure 4.

Classification of Plasmodium vivax recurrent infections with PvAmpSeq using IBD. (A-B). Distribution of IBD estimates in paired infections from the Solomon Islands and Peru, comparing baseline and recurrent infection pairs. (C-D). IBD between infection pairs in the Solomon Islands and Peru. The X-axis shows the days since baseline or first infection, the Y-axis depicts each patient’s infection over time, and the colour represents the IBD estimate range for the infection pairs. Baseline and first infections (Day 0) are not displayed for easier interpretation of plots.

Figure 5.

Model-based classification of recurrent P. vivax infections with PvAmpSeq. Marginal posterior probability estimates of classification outcome (relapse, recrudescence, or reinfection) of recurrent infections compared to the baseline infection faceted by each participant in (A) the Solomon Islands and (B) Peru. The X-axis shows the episode number; for example, considering that the baseline infection is the first episode, the first recurrent infection would be episode 2.