Piperaquine resistant Cambodian Plasmodium falciparum clinical isolates: in vitro genotypic and phenotypic characterization

Abstract

Background: High rates of dihydroartemisinin-piperaquine (DHA-PPQ) treatment failures have been documented for uncomplicated Plasmodium falciparum in Cambodia. The genetic markers plasmepsin 2 (pfpm2), exonuclease (pfexo) and chloroquine resistance transporter (pfcrt) genes are associated with PPQ resistance and are used for monitoring the prevalence of drug resistance and guiding malaria drug treatment policy.

Methods: To examine the relative contribution of each marker to PPQ resistance, in vitro culture and the PPQ survival assay were performed on seventeen P. falciparum isolates from northern Cambodia, and the presence of E415G-Exo and pfcrt mutations (T93S, H97Y, F145I, I218F, M343L, C350R, and G353V) as well as pfpm2 copy number polymorphisms were determined. Parasites were then cloned by limiting dilution and the cloned parasites were tested for drug susceptibility. Isobolographic analysis of several drug combinations for standard clones and newly cloned P. falciparum Cambodian isolates was also determined.

Results: The characterization of culture-adapted isolates revealed that the presence of novel pfcrt mutations (T93S, H97Y, F145I, and I218F) with E415G-Exo mutation can confer PPQ-resistance, in the absence of pfpm2 amplification. In vitro testing of PPQ resistant parasites demonstrated a bimodal dose-response, the existence of a swollen digestive vacuole phenotype, and an increased susceptibility to quinine, chloroquine, mefloquine and lumefantrine. To further characterize drug sensitivity, parental parasites were cloned in which a clonal line, 14-B5, was identified as sensitive to artemisinin and piperaquine, but resistant to chloroquine. Assessment of the clone against a panel of drug combinations revealed antagonistic activity for six different drug combinations. However, mefloquine-proguanil and atovaquone-proguanil combinations revealed synergistic antimalarial activity.

Conclusions: Surveillance for PPQ resistance in regions relying on DHA-PPQ as the first-line treatment is dependent on the monitoring of molecular markers of drug resistance. P. falciparum harbouring novel pfcrt mutations with E415G-exo mutations displayed PPQ resistant phenotype. The presence of pfpm2 amplification was not required to render parasites PPQ resistant suggesting that the increase in pfpm2 copy number alone is not the sole modulator of PPQ resistance. Genetic background of circulating field isolates appear to play a role in drug susceptibility and biological responses induced by drug combinations. The use of latest field isolates may be necessary for assessment of relevant drug combinations against P. falciparum strains and when down-selecting novel drug candidates.

Keywords: Drug combination; Exonuclease; Malaria; PfCRT; Piperaquine resistance; Plasmepsin.

Fig. 1

Survival assay for ART- and PPQ-resistance. a In vitro RSA_0-3h survival rates for standard laboratory-adapted clones (W2 for an ART-sensitive control, IPC-4884 and IPC-5202 for ART-resistance control) and culture-adapted clinical isolates. The dashed line represents the 1% survival rate cut-off that differentiates ART-resistance (≥ 1%, red-dashed line) from ART-sensitive (< 1%) parasites in RSAs. b In vitro PPQ survival assay (PSA_0–3 h) survival rates for standard laboratory-adapted clones (W2, IPC-4884 and IPC-5202 for PPQ-sensitive parasites) and culture-adapted clinical isolates. The dashed line represent the 10% survival rate cut-off that distinguishes PPQ-resistance (≥ 10%) from PPQ-sensitive (< 10%, red dashed line) parasites in PSAs. Two biological replicates were performed and survival rates are presented as mean ± S.D. Significance was determined using Mann–Whitney U test. Group 3 and Group 4 are compared. ns is not significant (p ≥ 0.05). Zero values of % survival rate were plotted as 0.001% in logarithmic scale

Fig. 2

In vitro P. falciparum susceptibility to multiple antimalarial drugs. Mean ± S.D IC₅₀ values were calculated from 72-h dose–response assays for drugs designated in a–i. White bars represent standard laboratory-adapted clones, while blue and red bars indicate clinical-adapted parasites with PPQ sensitive or PPQ resistance, respectively. Three biological replicates were carried out for each sample. Statistically significant differences relative to isolate 17 are indicated with one (0.05 > p > 0.01) and two (p < 0.01) asterisks

Fig. 3

PPQ dose–response curves and cell morphology for selected culture-adapted clinical isolates. Increasing the starting concentration and number of data points (24 points) for HRP2 ELISA dose–response curve provided a bimodal distribution of parasite response to PPQ exposure for PPQ-resistant parasites. The PPQ-sensitive W2 parasite (black line) is shown alongside the culture-adapted clinical isolates. The PPQ-sensitive clinical adapted parasites are shown in blue lines, whereas the PPQ-resistant clinical adapted parasites are represented in red lines. Data are shown as mean values from three biological replicates with S.D for isolates. Cell morphology of selected culture-adapted clinical isolates shows that isolate number 9 revealed distended, translucent DV (the red arrow) similar to the engineered parasite with PfCRT variants [26]. The scale bar, 50 µm

Fig. 4

Drug susceptibility of P. falciparum Cambodian isolate 14 before and after cloning against DHA, MQ, CQ, and PPQ. The W2 and 3D7 strains of P. falciparum were used as controls. Statistically significant differences relative to 3D7 and isolate 14 are indicated in black and red asterisks, respectively with 0.05 > p > 0.01 for one asterisk and p < 0.01 for two asterisks. Abbreviation, ns is for not significant difference (p ≥ 0.05)