Prevalence of Plasmodium falciparum drug resistance markers pfcrt K76T and pfaat1 S258L in southern Rwanda, 2010 to 2023.

Background: In many countries in Sub-Saharan Africa, the Plasmodium falciparum chloroquine resistance marker pfcrt K76T disappeared within a decade of ceased chloroquine use. Pfaat1 S258L has recently been implicated as another chloroquine resistance marker. Both genes may affect parasite susceptibility to partner drugs in artemisinin-based combination therapy. Rwanda abolished chloroquine in 2001, since 2006 the first-line antimalarial is artemether-lumefantrine. However, partial artemisinin resistance emerged in the region. We assessed the prevalence of pfcrt and pfaat1 markers in Huye district between 2010 -2023, following trends and updating the status in southern Rwanda.

Methods: P. falciparum positive blood samples from community children and malaria patients collected 2010, 2014, 2018, 2019 and 2023 were examined. Pfcrt K76T was genotyped by RFLP, pfaat1 S258L by high-resolution melting-curve (2010-2019). Samples from 2023 were subjected to nanopore sequencing.

Results: In 606 samples, pfcrt K76T prevalence declined from 76% (95% confidence interval, 68-83%) to 18% (11-25%) between 2010 and 2018 but stagnated since around 25% (P < 0.001). No further pfcrt markers were observed. Pfaat1 S258L remained at or near fixation. The artemisinin resistance marker pfk13 R561H was associated with pfcrt K76T (P = 0.02).

Discussion: The persistence of pfcrt K76T 20 years after abolishing chloroquine indicates ongoing drug selection or importation. The fixation of pfaat1 S258L argues against a major fitness cost of this variant in Huye. Partial artemisinin resistance increases in Rwanda, and molecular markers indicate compromised lumefantrine efficacy. The observed pfcrt and pfaat1 signatures in the study area might guide artemisinin partner drug alternatives.