The lack of K13-propeller mutations associated with artemisinin resistance in Plasmodium falciparum in Democratic Republic of Congo (DRC)

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 wildtype 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.

Biennial surveillance of Plasmodium falciparum anti-malarial drug resistance markers in Democratic Republic of Congo, 2017 and 2019

“…Samples with PCR-confirmed presence of P. falciparum were used to amplify segments of interest on pfk13 and pfcrt genes. The procedures previously described were used to amplify the interest segment on pfcrt gene [ 25 ] and that on the propeller region of pfk13 gene [ 23 ]. The analysis of P. falciparum genes polymorphisms was done by DNA bi-directional sequencing using Sanger method as previously described [ 15 , 23 ].…”

“…To help improve long-term monitoring of anti-malarial drug resistance, the present study aimed to provide baseline data for biennial molecular surveillance of P. falciparum anti-malarial drug resistance by comparing data from the study conducted in 2019 to previously published data from a similar study conducted in 2017 in the DRC [ 15 , 23 ].…”

Assessment of Plasmodium falciparum anti-malarial drug resistance markers in pfk13-propeller, pfcrt and pfmdr1 genes in isolates from treatment failure patients in Democratic Republic of Congo, 2018–2019

“…Although none of the mutations associated with ART resistance in Southeast Asia have been found in this study, 7 coding substitutions that are of unknown phenotype were observed, among which 3 have been previously reported, notably N498I in Kenya [21], N554K in Comoros and A557S in Togo and DRC [22], and 4 others that had not yet been reported (F506L, E507V, D516E, G538S). Numerous pfk13-propeller mutations of unknown function are commonly reported in sub-Saharan African countries, including the DRC [18,22,23]. There are criteria for prioritizing further laboratory studies, notably the frequent observation of a new allele with a non-synonymous mutation, the evidence of dissemination and preliminary association with clinical data whenever possible [23].…”

“…The target sequence was a 506-nucleotide fragment of the pfk13-propeller gene covering codon-positions 427-595, following a procedure previously described [18]. This segment of interest includes mutations associated with ART resistance [7].…”

How Physiologic Targets Can Be Distinguished from Drug-Binding Proteins

“…In isolated cases, reduced cell susceptibility to a drug against a metabolic enzyme occurs by mutations in the enzyme itself (Dicker et al, 1993;Wilson et al, 1992;Yun et al, 2008). However, the myriad of pathways employed by cells to counter susceptibility to drugs, including efflux and rewiring of metabolic pathways, is astounding (Aouida et al, 2004;Barrett et al, 2011;Chang et al, 2019;Chitanga et al, 2011;Claus et al, 2014;Dermawan et al, 2014;Gatti et al, 2015;Haider et al, 2020;Seyhan et al, 2012;Yobi et al, 2020;Zhang et al, 2019b). For these reasons, it is prudent to delay conclusions that genes identified by sequencing of genomes from drug-resistant cells are targets of the drugs until confirmatory data from other studies are obtained.…”