Abstract
Artemisinin-based combination therapies (ACTs) have been recommended by the World Health Organization (WHO) as first-line treatment of uncomplicated Plasmodium falciparum (P. falciparum) malaria since 2005 in Democratic Republic of Congo (DRC) and a regular surveillance of the ACT efficacy is required to ensure the treatment effectiveness. Mutations in the propeller domain of the pfk13 gene were identified as molecular markers of artemisinin resistance (ART-R). This study investigated the pfk13-propeller gene polymorphism in clinical isolates of P. falciparum collected in the DRC. In 2017, ten geographical sites across DRC were selected for a cross-sectional study that was conducted first in Kinshasa from January to March, then in the nine other sites from September to December. Dried blood samples were collected from patients attending health centers for fever where diagnosis of Malaria was first made by rapid diagnostic test (RDT) available on site (SD Bioline malaria Ag Pf or CareStart Malaria Pf) or by thick blood smear and then confirmed by a P. falciparum real-time PCR assay. A pfk13-propeller segment containing a fragment that codes for amino acids at positions 427-595 was amplified by conventional PCR before sequencing. In total, 1070 patients were enrolled in the study. Real-time PCR performed confirmed the initial diagnosis of P. falciparum infection in 806 samples (75.3%; 95% CI: 72.6%- 77.9%). Of the 717 successfully sequenced P. falciparum isolates, 710 (99.0%; 95% CI: 97.9% - 99.6) were wild-type genotypes and 7 (1.0%; 95% CI: 0.4% - 2.1%) carried non-synonymous (NS) mutations in pfk13-propeller including 2 mutations (A578S and V534A) previously detected and 2 other (M472I and A569T) not yet detected in the DRC. Mutations associated with ART-R in Southeast Asia were not observed in DRC. However, the presence of other mutations in pfk13-propeller gene calls for further investigations to assess their implication in drug resistance.
Highlights
Plasmodium falciparum (P. falciparum) is the most widespread Plasmodium species and is responsible for the majority of severe forms and deaths related to malaria in sub-Saharan Africa
Real-time PCR analysis performed on DNA extracted from Dried blood spots (DBS) confirmed the initial diagnosis of P. falciparum infection for 806 (75.3%; 95% confidence intervals (95% CI): 72.6%– 77.9%) patients
Of the 717 successfully sequenced P. falciparum isolates, 710 (99.0%; 95% CI: 97.9% - 99.6) were wildtype and 7 (1.0%; 95% CI: 0.4% - 2.1%) carried non-synonymous (NS) mutations in pfk13-propeller among which 2 previously described (A578S, V534A) and 2 mutations not yet described (M472I, A569T) in the Democratic Republic of Congo (DRC)
Summary
Plasmodium falciparum (P. falciparum) is the most widespread Plasmodium species and is responsible for the majority of severe forms and deaths related to malaria in sub-Saharan Africa. Mutations in the propeller domain of P. falciparum Kelch 13 gene (pfk13) have been identified as associated with in-vivo delayed parasite clearance and in-vitro artemisinin resistance (ART-R) in ring stage survival Assay (RSA) [4, 5]. These mutations spread into the Greater Mekong Sub-region (GMS) of Southeast Asia [6, 7] and have been recently classified in validated markers of ART-R (F446I, N458Y, M476I, Y493H, R539T, I543T, P553L, R561H and C580Y) and candidate/associated markers of ART-R (P441L, G449A, C469F, A481V, P527H, N537I, G538V, V568G, P574L, F673I, A675V) [8]. The spread of ART-R from the GMS to Africa, like previously happened with other antimalarial drugs [15, 16], may be a major obstacle for malaria control and elimination around the World
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