Distribution and Temporal Dynamics of Plasmodium falciparum Chloroquine Resistance Transporter Mutations Associated With Piperaquine Resistance in Northern Cambodia

Abstract

Background: Newly emerged mutations within the Plasmodium falciparum chloroquine resistance transporter (PfCRT) can confer piperaquine resistance in the absence of amplified plasmepsin II (pfpm2). In this study, we estimated the prevalence of co-circulating piperaquine resistance mutations in P. falciparum isolates collected in northern Cambodia from 2009 to 2017.

Methods: The sequence of pfcrt was determined for 410 P. falciparum isolates using PacBio amplicon sequencing or whole genome sequencing. Quantitative polymerase chain reaction was used to estimate pfpm2 and pfmdr1 copy number.

Results: Newly emerged PfCRT mutations increased in prevalence after the change to dihydroartemisinin-piperaquine in 2010, with >98% of parasites harboring these mutations by 2017. After 2014, the prevalence of PfCRT F145I declined, being outcompeted by parasites with less resistant, but more fit PfCRT alleles. After the change to artesunate-mefloquine, the prevalence of parasites with amplified pfpm2 decreased, with nearly half of piperaquine-resistant PfCRT mutants having single-copy pfpm2.

Conclusions: The large proportion of PfCRT mutants that lack pfpm2 amplification emphasizes the importance of including PfCRT mutations as part of molecular surveillance for piperaquine resistance in this region. Likewise, it is critical to monitor for amplified pfmdr1 in these PfCRT mutants, as increased mefloquine pressure could lead to mutants resistant to both drugs.

Keywords: Plasmodium falciparum; Cambodia; chloroquine resistance transporter; malaria; piperaquine resistance.

Figure 1.

Prevalence of newly emerged Plasmodium falciparum chloroquine resistance transporter (PfCRT) mutations in P. falciparum isolates collected from 2009 to 2017. Pie chart represents prevalence estimates from the complete data set (N = 410), regardless of year. The PfCRT protein schematic indicates the location of PfCRT mutations. Mutations observed in the Dd2 (green) and Cam734 (orange) haplotypes are shown at the top, while newly emerged PfCRT mutations (blue) are shown at the bottom.

Figure 2.

Ninety percent inhibitory concentrations (IC₉₀) to piperaquine in parasites with newly emerged Plasmodium falciparum chloroquine resistance transporter (PfCRT) mutations, with and without pfpm2 amplification. PfCRT mutations are shown on the x-axis, and piperaquine IC₉₀ is shown on the y-axis (log₁₀ scale). Blue box plots show the IC₉₀ distribution for parasites with single-copy pfpm2, while red box plots show the IC₉₀ distribution for parasites with amplified pfpm2. The “Any” category includes parasites with any of the following PfCRT mutations: F145I, H97Y, G353V, I218F, or T93S. The asterisked P value was estimated using a t test, while the other P values were estimated using linear regression, with the Dd2 haplotype serving as the reference. Only statistically significant P values (P < .05) are shown.

Figure 3.

Prevalence of antimalarial drug resistance mutations over time in Oddar Meanchey Province, Cambodia, 2009–2017. The green area represents the prevalence of amplified pfmdr1, while the gold and blue areas represent the prevalence of parasites with 2 or >2 copies of pfpm2, respectively. The dark green line indicates the prevalence of parasites harboring a Plasmodium falciparum chloroquine resistance transporter (PfCRT) mutation associated with piperaquine resistance, including T93S, H97Y, F145I, I218F, or G353V. Red arrows indicate years when the first-line drug treatment changed, with the switch to dihydroartemisinin-piperaquine occurring in 2010 and to artesunate-mefloquine in 2016.

Figure 4.

Prevalence of newly emerged Plasmodium falciparum chloroquine resistance transporter mutations over time in Oddar Meanchey Province, Cambodia, 2009–2017. The area represents the proportion of isolates comprised of each mutation by year according to color. Mutations present in ≤2 isolates in the data set were not plotted. Red arrows indicate years when the first-line drug treatment changed, with the switch to dihydroartemisinin-piperaquine occurring in 2010 and to artesunate-mefloquine in 2016.