Expression of Plasmodium vivax crt-o Is Related to Parasite Stage but Not Ex Vivo Chloroquine Susceptibility

Abstract

Chloroquine (CQ)-resistant Plasmodium vivax is present in most countries where P. vivax infection is endemic, but the underlying molecular mechanisms responsible remain unknown. Increased expression of P. vivax crt-o (pvcrt-o) has been correlated with in vivo CQ resistance in an area with low-grade resistance. We assessed pvcrt-o expression in isolates from Papua (Indonesia), where P. vivax is highly CQ resistant. Ex vivo drug susceptibilities to CQ, amodiaquine, piperaquine, mefloquine, and artesunate were determined using a modified schizont maturation assay. Expression levels of pvcrt-o were measured using a novel real-time quantitative reverse transcription-PCR method. Large variations in pvcrt-o expression were observed across the 51 isolates evaluated, with the fold change in expression level ranging from 0.01 to 59 relative to that seen with the P. vivax β-tubulin gene and from 0.01 to 24 relative to that seen with the P. vivax aldolase gene. Expression was significantly higher in isolates with the majority of parasites at the ring stage of development (median fold change, 1.7) compared to those at the trophozoite stage (median fold change, 0.5; P < 0.001). Twenty-nine isolates fulfilled the criteria for ex vivo drug susceptibility testing and showed high variability in CQ responses (median, 107.9 [range, 6.5 to 345.7] nM). After controlling for the parasite stage, we found that pvcrt-o expression levels did not correlate with the ex vivo response to CQ or with that to any of the other antimalarials tested. Our results highlight the importance of development-stage composition for measuring pvcrt-o expression and suggest that pvcrt-o transcription is not a primary determinant of ex vivo drug susceptibility. A comprehensive transcriptomic approach is warranted for an in-depth investigation of the role of gene expression levels and P. vivax drug resistance.

Measuring ex vivo drug susceptibility in Plasmodium vivax isolates from Cambodia

“…Comparing findings of the present study to those reported previously for P. falciparum and P. vivax isolates from the Brazilian Amazon [ 46 , 47 ] and Indonesia [ 36 , 48 , 49 ] using pLDH ELISA and SMT assays, Cambodian isolates were found to be less susceptible to MQ and PPQ. However, Cambodian and Brazillian P. vivax isolates were more sensitive to CQ than in Papua Indonesia where P. vivax CQ resistance has emerged.…”

Selective sweep suggests transcriptional regulation may underlie Plasmodium vivax resilience to malaria control measures in Cambodia

“…Histone deacetylase inhibitors accelerated the rate of hypnozoite activation in long-term primary simian hepatocyte cultures, suggesting that histone methylation may maintain hypnozoite dormancy by suppressing transcription (42,43). Finally, P. vivax chloroquine resistance has not been found to correlate with DNA changes in pvcrt but may correlate with the expression of this transporter protein, whose ortholog mediates chloroquine resistance in P. falciparum (44)(45)(46).…”

Plasma metabolomics reveals membrane lipids, aspartate/asparagine and nucleotide metabolism pathway differences associated with chloroquine resistance in Plasmodium vivax malaria

“…In terms of gene transcription, however, parasites from patients with PvCR presented up to 6.1 and 2.4 fold increase in pvcrt-o and pvmdr-1 transcription levels, respectively, compared to the susceptible group [ 26 ]. Caution is needed when attempting to extrapolate the ex vivo biomarkers of resistance to the clinical response, since pvcrt-o transcription was not a primary determinant of ex vivo drug susceptibility [ 77 ]. These previous observations suggest that host-parasite interaction factors, such as nutritional and immune components and clinical severity grading, rather than parasite constitutional factors per se , may be involved in PvCR [ 26 , 27 , 78 ].…”