Hypnozoite depletion in successive Plasmodium vivax relapses

Abstract

Genotyping Plasmodium vivax relapses can provide insights into hypnozoite biology. We performed targeted amplicon sequencing of 127 relapses occurring in Indonesian soldiers returning to malaria-free Java after yearlong deployment in malarious Eastern Indonesia. Hepatic carriage of multiple hypnozoite clones was evident in three-quarters of soldiers with two successive relapses, yet the majority of relapse episodes only displayed one clonal population. The number of clones detected in relapse episodes decreased over time and through successive relapses, especially in individuals who received hypnozoiticidal therapy. Interrogating the multiplicity of infection in this P. vivax relapse cohort reveals evidence of independent activation and slow depletion of hypnozoites over many months by multiple possible mechanisms, including parasite senescence and host immunity.

Fig 1. Genotypes of 33 pairs of relapses in returning Indonesian soldiers based on pvmsp1 amplicon deep sequencing.

The in-host frequency of different pvmsp1 haplotype variants is displayed for the first (-1) and second relapse (-2) in each individual. 17 pairs showed a homologous relapse pattern (A), 5 demonstrated minor variant expansion (B), and 11 were heterologous relapses (C). Though 61% of relapses were monoclonal, only 24% had a single variant detected across both relapses. Examples of each relapse pattern are shown in (D).

Fig 2. Decreased clonal burden in relapses over time.

(A) Among 127 relapses in returning Indonesian soldiers, the number of pvmsp1 clones detected at relapse decreased over time, as measured by days since enrollment one month after arriving to Java (slope = -0.003, p = 0.01). (B) Among 33 Indonesian soldiers with dual relapse, the multiplicity of infection (MOI) decreased between the first and second relapse episode, driven by those who received primaquine at their first relapse (depicted in blue) (slope = -.007, p = 0.03). Similarly, relapses in 18 Cambodian soldiers determined genotypically to have relapse [13] suggest a decrease in MOI from their initial vivax episode to relapse (slope = -0.03, p = 0.20). Lines of best fit were determined by linear regression.