Impact of piperaquine resistance in Plasmodium falciparum on malaria treatment effectiveness in The Guianas: a descriptive epidemiological study

Abstract

Background: Plasmodium falciparum is an apicomplexan parasite responsible for lethal cases of malaria. According to WHO recommendations, P falciparum cases are treated with artemisinin-based combination therapy including dihydroartemisinin-piperaquine. However, the emergence of resistant parasites against dihydroartemisinin-piperaquine was reported in southeast Asia in 2008 and, a few years later, suspected in South America.

Methods: To characterise resistance emergence, a treatment efficacy study was performed on the reported patients infected with P falciparum and treated with dihydroartemisinin-piperaquine in French Guiana (n=6, 2016-18). Contemporary isolates collected in French Guiana were genotyped for P falciparum chloroquine resistance transporter (pfCRT; n=845) and pfpm2 and pfpm3 copy number (n=231), phenotyped using the in vitro piperaquine survival assay (n=86), and analysed through genomic studies (n=50). Additional samples from five Amazonian countries and one outside the region were genotyped (n=1440).

Findings: In field isolates, 40 (47%) of 86 (95% CI 35·9-57·1) were resistant to piperaquine in vitro; these phenotypes were more associated with pfCRT^C350R (ie, Cys350Arg) and pfpm2 and pfpm3 amplifications (Dunn test, p<0·001). Those markers were also associated with dihydroartemisinin-piperaquine treatment failure (n=3 [50%] of 6). A high prevalence of piperaquine resistance markers was observed in Suriname in 19 (83%) of 35 isolates and in Guyana in 579 (73%) of 791 isolates. The pfCRT^C350R mutation emerged before pfpm2 and pfpm3 amplification in a temporal sequence different from southeast Asia, and in the absence of artemisinin partial resistance, suggesting a geographically distinctive epistatic relationship between these genetic markers.

Interpretation: The high prevalence of piperaquine resistance markers in parasite populations of the Guianas, and the risk of associated therapeutic failures calls for caution on dihydroartemisinin-piperaquine use in the region. Furthermore, greater attention should be given to potential differences in genotype to phenotype mapping across genetically distinct parasite populations from different continents.

Funding: Pan American Health Organization and WHO, French Ministry for Research, European Commission, Santé publique France, Agence Nationale de la Recherche, Fundação de Amparo à Pesquisa do Estado do Amazonas, Ministry of Health of Brazil, Oswaldo Cruz Foundation, and National Institutes of Health.

Translations: For the French and Portuguese translations of the abstract see Supplementary Materials section.

Figure 1

Piperaquine resistance and pfCRT^C350 allele status in French Guiana, 1997–2018 (A) The pfCRT^C350R mutation is associated with higher parasite survival rates after 48 h of piperaquine exposure (piperaquine survival assay). The dashed line marks the resistance threshold for piperaquine. (B) The pfCRT^C350R mutation was first detected in 2002, and then rapidly increased in frequency. Between 2005 and 2006, the prevalence of this mutation rose from 2 (8%) of 25 patients (95% CI 0–18·9%) to 15 (40%) of 38 (23·9–55·0%). Since 2008, pfCRT^C350R isolates have accounted for more than half of collected isolates each year. The blue bars represent pfCRT^C350 wild-type isolates and the red bars, pfCRT^C350R mutants. Error bars represent the 95% CIs. pfCRT^C350R=plasmodium falciparum chloroquine resistance transporter (ie, Cys350Arg). ***p<0·001.

Figure 2

Dihydroartemisinin–piperaquine therapeutics that did not work, reported in French Guiana, 2016–18 Parasitaemia was calculated as: percent infected red blood cells=(number of infected red blood cells ÷ total number of red blood cells counted) × 100 or, positive PCR=positive results after real-time PCR falciparum-specific exhibiting a sensitivity of 1 parasite per μl of blood. Plasmatic drug dosages were evaluated by high-performance liquid chromatography with diode array; however dihydroartemisinin–piperaquine administration was supervised. The number of days elapsed from the beginning of the treatment is shown on the x-axis. Correct piperaquine intake was confirmed if plasma concentration was ≥30 ng/ml after day 7. No patients returned to endemic areas during or after treatment. Monoclonality and recrudescence of parasite was confirmed by PCR genotyping the pfmsp1, pfmsp2, and pfglurp alleles in samples at day 0 and the day the treatment was deemed to have not worked. Survival assays under dihydroartemisinin or piperaquine pressure (700 nM and 200 nM, respectively) defined the survival rate of the parasites in vitro. Red text boxes detail genetic markers associated with parasite resistance phenotypes and black text boxes for sensitive phenotypes. Piperaquine resistance markers are highlighted in purple. Following treatment that did not work, patients recovered after atovaquone–proguanil treatment. Pfk13 WT defines a genotype on the propeller region identical to the reference strain 3D7. Pfpm2 and pfpm3=plasmepsin 2 and the plasmepsin 3. PfCRT=Plasmodium falciparum chloroquine resistance transporter.

Figure 3

Whole-genome sequencing of 50 phenotyped Plasmodium falciparum isolates (A) A genome-wide association analysis with 4600 single nucleotide polymorphisms shows a single outlier at the nucleotide position encoding the Cys350Arg substitution in the pfCRT³⁵⁰ locus (red). The dashed line shows the α=0·05 significance level after multiple test corrections. The mutation shows evidence of carrying a large phenotypic effect, as the effect size estimate (β in the generalised linear mixed model) was 9·8 (SE 1·7) with percent survival in the piperaquine survival assay ranging from 0% to 45% for parasites with the mutation. (B) Mean relatedness (identity-by-descent) between pairs of individuals calculated in 50 kb windows across the genome shows high variability genome wide. The genomic region surrounding the pfCRT locus (red line) does not show elevated relatedness, indicative of a soft selective sweep. Dashed grey lines depict the 50th and 95th percentiles. (C) The whole-genome sequenced samples of French Guiana origin (n=50) harbour 12 unique haplotypes in the 200 kb region surrounding the pfCRT³⁵⁰ locus (chromosome 7, positions 305 000 to 505 000). Each row corresponds to a single sample. Each column corresponds to a variant site. Blue denotes the 3D7 reference allele. Orange denotes the alternate allele. Red marks the mutation coding for pfCRT^C350R. Three of these haplotypes contain both wild-type and mutant pfCRT³⁵⁰ alleles. PfCRT=plasmodium falciparum chloroquine resistance transporter.

Figure 4

Distribution of piperaquine resistance markers from isolates collected between 1997 and 2018 and spatial distribution of Plasmodium falciparum malaria infections in 2017 (A) Frequencies of isolates with pfCRT muted (red bars) or not (blue bars) at position 350 showed disparities between the countries in the Amazon basin (isolate population=n). (B) Spatial distribution of piperaquine resistance markers (pfCRT^C350R [ie, Cys350Arg] and pfpm2 and pfpm3 amplification) in Guiana Shield countries from isolates collected between 2006 and 2018. Blue designated for pfCRT^C350 carriers with a single copy of pfpm2 and pfpm3, light blue for pfCRT^C350 carriers with multiple copies of pfpm2 and pfpm3, red for pfCRT^C350R mutants with a single copy of pfpm2 and pfpm3, and pink for pfCRT^C350R mutants with multiple copies of pfpm2 and pfpm3. Adapted from annual country reports to the Pan American Health Organization, CDE/VT, and Malaria.Pfpm2 and Pfpm3=plasmepsin 2 and plasmepsin 3. PfCRT=Plasmodium falciparum chloroquine resistance transporter.

Figure 5

Emergence and impact of plasmepsin gene amplifications (pfpm2 and pfpm3) in French Guianese isolates between 1997 and 2018 (A) Prevalence of pfCRT³⁵⁰ polymorphism (isolate population=N) and pfpm2 and pfpm3 multiple copies (isolate population=n). Like pfCRT^C350R (ie, Cys350Arg), pfpm2 and pfpm3 amplifications rapidly rose in frequency after their initial emergence, culminating in 29 (83%) of 35 isolates (95% CI 70·4–95·3%) between 2012 and 2014. Blue bars represent pfCRT^C350 carriers and red bars, pfCRT^C350R mutants; black error bars show the SD between the years. The green dashed line shows the percentage of isolates with multiple copies of pfpm2 and pfpm3, green error bars display the 95% CI. (B) The pfpm2 and pfpm3 copy number status depending on the pfCRT³⁵⁰ polymorphism (isolate population=n). Blue bars represente pfCRT^C350 carriers with a single copy of pfpm2 and pfpm3, light-blue bars for pfCRT^C350 carriers with multiple copies of pfpm2 and pfpm3, red bars for pfCRT^C350R mutants with a single copy of pfpm2 and pfpm3, and pink bars for pfCRT^C350R mutants with multiple copies of pfpm2 and pfpm3. (C) Survival rate after piperaquine pressure depending on isolate genotype. Blue circles represent pfCRT^C350 carriers with a single copy of pfpm2 and pfpm3, light-blue circles indicate pfCRT^C350 carriers with multiple copies of pfpm2 and pfpm3, red circle indicate pfCRT^C350R mutants with a single copy of pfpm2 and pfpm3, and pink circles indicate of pfCRT^C350R mutants with multiple copies of pfpm2 and pfpm3. Multiple copies of genes is ≥2. Significance was determined by a Dunn test for multiple comparisons using R; *p<0·05, **p<0·01, and ***p<0·001. Sample size=n. (D) Temporal analysis of piperaquine resistance markers emergence and prevalence in southeast Asia and South America. PfCRT mutation is in red and pfpm2 and pfpm3 amplifications are in green. The timeline depicts periods from 2000 to 2020. Coloured arrows pinpoint the time of the first emergence for each piperaquine resistance markers. The different drug pressures are represented, with dashed arrows for non-conventional usage. Pfpm2 and pfpm3=plasmepsin 2 and the plasmepsin 3. PfCRT=Plasmodium falciparum chloroquine resistance transporter.