Molecular surveillance of anti-malarial drug resistance in Democratic Republic of Congo: high variability of chloroquinoresistance and lack of amodiaquinoresistance

Abstract

Background: The loss of chloroquine (CQ) effectiveness has led to its withdrawal from national policies as a first-line treatment for uncomplicated malaria in several endemic countries, such as the Democratic Republic of Congo (DRC). The K76T mutation on the pfcrt gene has been identified as a marker of CQ resistance and the SVMNT haplotype in codons 72-76 on the same gene has been associated with resistance to amodiaquine (AQ). In the DRC, the prevalence of K76T has decreased from 100% in 2000 to 63.9% in 2014. The purpose of this study was to determine the prevalence of K76T mutations in circulating strains of Plasmodium falciparum, 16 years after CQ withdrawal in the DRC and to investigate the presence of the SVMNT haplotype. Methods: In 2017, ten geographical sites across the DRC were selected. Dried blood samples were collected from patients attending health centres. Malaria was first detected by a rapid diagnostic test (RDT) available on site (SD Bioline Malaria Ag Pf or CareStart Malaria Pf) or thick blood smear and then confirmed by a P. falciparum species-specific real-time PCR assay. A pfcrt gene segment containing a fragment that encodes amino acids at positions 72-76 was amplified by conventional PCR before sequencing. Results: A total of 1070 patients were enrolled. Of the 806 PCR-confirmed P. falciparum positive samples, 764 were successfully sequenced. The K76T mutation was detected in 218 samples (28.5%; 95% CI 25.4%-31.9%), mainly (96%) with the CVIET haplotype. Prevalence of CQ resistance marker was unequally distributed across the country, ranging from 1.5% in Fungurume to 89.5% in Katana. The SVMNT haplotype, related to AQ resistance, was not detected. Conclusion: Overall, the frequency of the P. falciparum CQ resistance marker has decreased significantly and no resistance marker to AQ was detected in the DRC in 2017. However, the between regions variability of CQ resistance remains high in the country. Further studies are needed for continuous monitoring of the CQ resistance level for its prospective re-use in malaria management. The absence of the AQ resistance marker is in line with the use of this drug in the current DRC malaria treatment policy.

Surveillance of Plasmodium falciparum pfcrt haplotypes in southwestern Uganda by high‐resolution melt analysis

“…The prevalence of CQ resistance haplotypes was assessed in parasite populations from multiple sites across Southwestern Uganda (Ibanda, Isingiro, Kiruhura, and Mbarara collected in 2010, and Kasese collected in 2015). Dominance of the mutant CVIET haplotype in the samples, and lack of the mutant SVMNT haplotype (Table 1), is consistent with what has been observed in other areas of Uganda and throughout sub-Saharan Africa during a similar time frame [5,8,20,21,26,27,40,44,54]. Additionally, the prevalence of CVIET in Group 2 samples (~ 45 %, from Kasese, Table 1) is consistent with previous findings in the region (~ 50 %, from Kanungu in 2016, [28], see Fig.…”

“…Molecular genotyping studies have predominantly used gene-specific PCR followed by sequencing to identify resistance conferring mutations [26,27,44]. Other approaches such as ligase detection reaction-fluorescent microsphere assay [28,40,45] and the quantitative PCRbased malariaTAC [46] facilitate higher sample throughput and the parallel assessment of multiple resistance alleles.…”

“…In other regions of the world, CQ resistant parasites persist decades after CQ cessation [17][18][19]. In general, a multitude of studies across Africa have observed a wide range in the proportion of CQ resistant parasites [20][21][22][23][24][25][26][27][28], which emphasizes the need for continued surveillance of resistance markers.…”

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

“…The global prevalence of the K76T mutation known to be associated with CQ resistance was 41.5%, but this was variable from one site to another, ranging from 10.0% in Fungurume to 76.9% in Katana. In 2017, a study conducted in 10 sites (including 6 sites of the present study) reported a global prevalence of the K76T mutation of 28.5% but with a high between-regions variability ranging from 1.5% in Fungurume to 89.5% in Katana [27]. In the present study, the prevalence of K76T in patients treated with ASAQ (48.9%) was higher than in those treated with AL (37.4%) with low statistical difference (p value = 0.038).…”

“…The SVMNT haplotype associated with AQ resistance was not detected in the present study, which is encouraging for the DRC national policy for the continued use of AQ in ACT. This haplotype has not yet been reported in the DRC [19,26,27,30] whereas it was found in neighbouring countries, such as Tanzania and Angola [31,32].…”