Multiple-Clone Activation of Hypnozoites Is the Leading Cause of Relapse in <em>Plasmodium vivax</em> Infection

<div><h3>Background</h3><p><em>Plasmodium vivax</em> infection is characterized by a dormant hepatic stage, the hypnozoite that is activated at varying periods of time after clearance of the primary acute blood-stage, resulting in relapse. Differentiation between treatment failure and new infections requires characterization of initial infections, relapses, and clone multiplicity in vivax malaria infections.</p> <h3>Methodology/Principal Findings</h3><p>Parasite DNA obtained from primary/relapse paired blood samples of 30 patients with <em>P. vivax</em> infection in Brazil was analyzed using 10 molecular markers (8 microsatellites and MSP-1 blocks 2 and 10). Cloning of PCR products and genotyping was used to identify low-frequency clones of parasites. We demonstrated a high frequency of multiple-clone infections in both primary and relapse infections. Few alleles were identified per locus, but the combination of these alleles produced many haplotypes. Consequently, the majority of parasites involved in relapse showed haplotypes that were distinct from those of primary infections. <em>Plasmodium vivax</em> relapse was characterized by temporal variations in the predominant parasite clones.</p> <h3>Conclusions/Significance</h3><p>The high rate of low frequency alleles observed in both primary and relapse infections, along with temporal variation in the predominant alleles, might be the source of reported heterologous hypnozoite activation. Our findings complicate the concept of heterologous activation, suggesting the involvement of undetermined mechanisms based on host or environmental factors in the simultaneous activation of multiple clones of hypnozoites.</p> </div

Detection of multiple-clone <i>P. vivax</i> infections using a panel of 10 markers.

<p>(A) Number and percent of malaria episodes showing multiple-clone infections detected by different numbers of markers. (B) Minimum number of markers able to detect all multiple clone infections was five: MS01 (77%), MS01+ MS07 (92%), MS01+ MS07+ MSPBl2 (97%), MS01+ MS07+ MSPBl2+ MS11 (98%), MS01+ MS07+ MSPBl2+ MS11+ MS08 (100%).</p

Genotyping of <i>P. vivax</i> primary/relapse paired parasites from 30 patients using 10 molecular markers.

<p>(A) Haplotype derived from predominant allele of each marker. Totally homologous – parasites showing all markers with the same allele; Homologous or Related – parasites with 8 to 9 markers with the same allele; Heterologous – parasites showing less than 8 markers with the same allele (according to Orjuela-Sánchez et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049871#pone.0049871-OrjuelaSnchez1" target="_blank">[21]</a>). In patients with more than one relapse episodes, relapse parasites were compared with the previous acute malaria episode. (B) Percent of acute malaria episodes showing different amounts of markers with the same alleles, taking into account only the predominant allele from each marker (left) or all alleles, predominant and rare from each marker (right).</p

Percent of multiple-clone infections using different cut-off criteria.

<p>The detection of rare alleles in the genotyping was based on three cut-off criteria: ≥150 rFU (here); peaks with more than one quarter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049871#pone.0049871-Imwong3" target="_blank">[46]</a>; or with more than one third of the predominant peak height <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049871#pone.0049871-Gunawardena1" target="_blank">[47]</a>.</p

Genotypic profile before and after PCR cloning.

<p>PCR products from primary infection samples amplified using two randomly chosen molecular markers of four patients were cloned, and up to 26 colonies (mean of 11 colonies) were genotyped. (A) Each form represents an allele, size indicates predominant (larger) or rare alleles (smaller); color represents alleles of a distinct marker: MSP1bl10– red; MS07– blue; MSP1bl2– green; MS02– purple. The presence of two or more forms characterizes a multiple-clone infection. Before cloning the predominant allele was identified as the heighest peak in genotyping and the rare allele as the peak with one-third of the predominant peak height. After cloning the frequencies were inferred by the number of bacteria clones. The only relapse sample also cloned is indicated by an asterisk. (B) Frequency of each allele in primary infections after cloning measured by the percent of bacterial colonies genotyped with each allele (represented by different colors).</p

Temporal variation of the predominant alleles.

<p>(A) Comparison of predominant alleles among primary infection (PI), first relapse (R1), second relapse (R2), and third relapse (R3) from four <i>P. vivax</i>-infected patients genotyped using 10 molecular markers. Alleles are represented by different forms for each marker (indicated on the right side) and delimited by dotted lines. MS – microsatellite numbered according to Rezende et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049871#pone.0049871-Nishimura1" target="_blank">[36]</a>, MSP1bl2 and MSP1bl10– merozoite surface antigen 1 blocks 2 and 10, respectively. Haplotypes are indicated at the top of the Figure. (B) Frequencies of markers showing the same or distinct alleles at different times of blood collection for these four patients.</p