Impact of parasite genomic dynamics on the sensitivity of Plasmodium falciparum isolates to piperaquine and other antimalarial drugs

Abstract

Background: Dihydroartemisinin-piperaquine (DHA-PPQ) is an alternative first-line antimalarial to artemether-lumefantrine in Kenya. However, recent reports on the emergence of PPQ resistance in Southeast Asia threaten its continued use in Kenya and Africa. In line with the policy on continued deployment of DHA-PPQ, it is imperative to monitor the susceptibility of Kenyan parasites to PPQ and other antimalarials.

Methods: Parasite isolates collected between 2008 and 2021 from individuals with naturally acquired P. falciparum infections presenting with uncomplicated malaria were tested for in vitro susceptibility to piperaquine, dihydroartemisinin, lumefantrine, artemether, and chloroquine using the malaria SYBR Green I method. A subset of the 2019-2021 samples was further tested for ex vivo susceptibility to PPQ using piperaquine survival assay (PSA). Each isolate was also characterized for mutations associated with antimalarial resistance in Pfcrt, Pfmdr1, Pfpm2/3, Pfdhfr, and Pfdhps genes using real-time PCR and Agena MassARRAY platform. Associations between phenotype and genotype were also determined.

Results: The PPQ median IC₅₀ interquartile range (IQR) remained stable during the study period, 32.70 nM (IQR 20.2-45.6) in 2008 and 27.30 nM (IQR 6.9-52.8) in 2021 (P=0.1615). The median ex vivo piperaquine survival rate (IQR) was 0% (0-5.27) at 95% CI. Five isolates had a PSA survival rate of ≥10%, consistent with the range of PPQ-resistant parasites, though they lacked polymorphisms in Pfmdr1 and Plasmepsin genes. Lumefantrine and artemether median IC₅₀s rose significantly to 62.40 nM (IQR 26.9-100.8) (P = 0.0201); 7.00 nM (IQR 2.4-13.4) (P = 0.0021) in 2021 from 26.30 nM (IQR 5.1-64.3); and 2.70 nM (IQR 1.3-10.4) in 2008, respectively. Conversely, chloroquine median IC₅₀s decreased significantly to 10.30 nM (IQR 7.2-20.9) in 2021 from 15.30 nM (IQR 7.6-30.4) in 2008, coinciding with a decline in the prevalence of Pfcrt 76T allele over time (P = 0.0357). The proportions of piperaquine-resistant markers including Pfpm2/3 and Pfmdr1 did not vary significantly. A significant association was observed between PPQ IC₅₀ and Pfcrt K76T allele (P=0.0026).

Conclusions: Circulating Kenyan parasites have remained sensitive to PPQ and other antimalarials, though the response to artemether (ART) and lumefantrine (LM) is declining. This study forms a baseline for continued surveillance of current antimalarials for timely detection of resistance.

Keywords: Antimalarial; Artemisinin combined therapy; Drug resistance; Genomic; Plasmodium falciparum; Sensitivity.

Fig. 1

Map of Kenya depicting the six hospital surveillance sites

Fig. 2

Temporal changes of parasite’s response to antimalarials between 2008–2013, 2014–2017, and 2018–2021. Scatter plots with bars and whiskers showing median IC₅₀s and interquartile range (nM). P < 0.05 representative of statistically significant change in IC₅₀ represented by ** for chloroquine, lumefantrine, and artemether while the rest remained unchanged

Fig. 3

Scatter plot showing A ex vivo piperaquine survival rates of Kenyan clinical isolates from two locations and B susceptibility of parasites from six different geographic locations in Kenya to piperaquine using malaria SYBR green I assay. Horizontal bars and whiskers represent the median and interquartile range of susceptibility

Fig. 4

Association between drug resistance polymorphisms and in vitro susceptibility to piperaquine measured by SYBR green I assay. Scatter plots with horizontal bars and whiskers representing median IC₅₀s and interquartile ranges for each locus. M-mutant genotype, W- wild-type genotype, W/M- mixture of mutant and wild-type genotype in an infection. Pfcrt, Plasmodium falciparum chloroquine resistance gene; Pfmdr1, Plasmodium falciparum multidrug resistance gene 1; Pfdhps, Plasmodium falciparum dihydropteroate synthetase gene; Pfdhfr, Plasmodium falciparum dihydrofolate reductase gene; Pfpm2, Plasmodium falciparum plasmepsin 2 gene; and Pfpm3, Plasmodium falciparum plasmepsin 3 gene