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

The spread of resistance to anti-malarial drugs poses a serious public health risk and a real challenge for malaria control in endemic countries. As result of high treatment failure rate (> 10%) with chloroquine (CQ) and sulfadoxine–pyrimethamine (SP) and following the World Health Organization (WHO) recommendation, the Democratic republic of Congo (DRC) malaria national policy implemented artemisinin-based Combination Therapies (ACT) as first-line treatment of uncomplicated Plasmodium falciparum malaria in 2005 [1, 2]. WHO recommends biennial surveillance of anti-malarial drug efficacy to ensure early detection of emergence and spread of resistance. Therapeutic efficacy studies (TESs) are the gold standard for the monitoring of anti-malarial efficacy. However, today, in addition to TETs, the assessment of molecular marker in P. falciparum genes associated with anti-malarial drug resistance provides useful information about the emergence and spread of resistant parasites [3].

Point mutations in the chloroquine resistance transporter gene (pfcrt), notably mutations in amino acids position 72 to 76 have been found to confer resistance to both CQ and amodiaquine (AQ) [4]. The amino acid substitution from lysine (K) to threonine (T) at position 76 of the PFCRT protein (K76T) has been shown to confer CQ resistance [5], while the 72–76 PFCRT haplotype, namely SVMNT (Serine–Valine–Methionine–Asparagine–Threonine) has been linked to AQ resistance [6]. K76T mutation, which is considered as the most reliable molecular marker of CQ resistance, is found in all CQ-resistant isolates of P. falciparum [5]. The decline in this CQ-resistance marker following the official CQ removal from national treatment guidelines varies significantly between countries. In several countries, it was reported that after the discontinuance of CQ use, K76T-mutant parasites have been replaced by wild ones, resulting in the decrease in prevalence of K76T marker [7‐10]. In other countries, studies reported the persistence of resistant strains several years after stopping the use of CQ as first-line treatment of uncomplicated malaria [11, 12]. In the DRC, the prevalence of K76T marker was 55.4% in a study conducted with samples obtained from 2007 Demographic and Health Survey (2007-DHS) [13] and 63.9% in a study conducted in 2014 [14]. Moreover, a large variability in this prevalence of K76T was observed across the DRC in 2017, with presence of very high prevalence (89.5%) in one province and very low (1.5%) in another [15].

Single-nucleotide polymorphisms (SNPs) occurring within P. falciparum Kelch 13 gene (pfk13), precisely in the propeller domain region, have been described as molecular markers of ART resistance [16, 17]. The list of these SNPs categorized as validated mutations and candidate mutations of ART resistance is updated by the WHO [18]. Originally, these mutations have emerged and spread in Southeast Asia, but nowadays, some of them are increasingly detected in sub-Saharan African countries providing evidence for de novo emergence of resistance to ART in Sub-Saharan African countries [19‐22].

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].

Like in 2017-study [23], the 2019-study did not detect any polymorphisms that are currently known to be associated with resistance to ART. However, other coding substitutions observed, such as A578S, should be considered. A578S mutation, commonly reported in African countries including the DRC [23, 26, 27], was the only one which was detected in both 2019-study and 2017-study. Although, the amino acid substitution in A578S mutation is likely to alter the function of PFK13 protein [28], previous studies have not shown the link with ART resistance [17, 27]. The function of the pfk13 gene is largely unknown and the phenotypic expression of this gene could involve other determinants which could vary from one geographical region to another due to the genetic diversity of P falciparum. Other unusual polymorphisms have been observed by both the 2019-study and 2017-study; some of them (M472I, V534A and A569T) were previously reported in African countries [26]. Since the polymorphisms associated with ART resistance are numerous and occur on a background of genetic diversity, the mutations that are increasingly observed in Africa merit further characterization [29, 30].

Globally, the prevalence of the K76T mutation known to be associated with CQ-resistance continues to decline in the DRC. Indeed, compared to previously reported prevalence which was more than 50% [13, 14, 25], the present study has shown that the prevalence of CQ-resistance marker has decreased to less than 30% (28.5% in 2017-study and 22.7% in 2019-study) in the DRC. However, this prevalence remained very variable from one region to another, with the highest prevalence in Katana (89.5% in 2017-study and 84.2% in 2019-study) and the lowest in Fungurume (1.5% in 2017-study and 1.4% in 2019-study). A possible illicit use of CQ that contributes in maintaining CQ selective pressure on the local P. falciparum population could explain the persistence of high CQ resistance rate in some regions. In addition, the selection for CQ-resistance is largely dependent on the genetic structure of parasite populations, which have been shown to vary greatly between regions of the DRC [30]; this may partly explain the observed regional variability in prevalence of CQ-resistance rate. Other potential explanations for the heterogeneity in prevalence of CQ-resistance marker in different regions of the DRC have been addressed in the previous 2017-study [15].

The return of CQ-susceptible malaria may present a novel opportunity for reintroducing this molecule to prevent malaria, especially in vulnerable populations. Indeed, if the proportion of CQ-resistant parasites declines in African endemic countries to an undetectable level, the reintroduction of CQ to be used alone or in combination with other anti-malarial drugs for prophylaxis or treatment may possibly be considered. The absence of drug pressure is supposed to be the key driver in the reemergence of CQ-sensitivity parasites in the field setting [31]. Unfortunately, with the advent of the COVID-19 pandemic, CQ has been largely used for its supposed antiviral properties in affected countries including the DRC [32]. In this context, CQ has been made widely available in private drugstores with the presence of falsified version containing low amount of CQ, which was discovered in the DRC [33]. This is likely to maintain CQ pressure on P. falciparum populations, thus compromising the hope towards the return of CQ-susceptible malaria in endemic countries particularly in the DRC.

The PFCRT 72–76 haplotype, SVMNT has been correlated with high-level resistance to the AQ metabolite desethylamodiaquine (DEAQ) in in vitro tests [34]. Like in 2017-study, the SVMNT haplotype associated with AQ-resistance was not detected in 2019-study. AQ is one of the partner drugs of ACTs used in many endemic countries like the DRC for the treatment of uncomplicated P. falciparum malaria. Generally, AQ was found to be effective in Africa, even in the face of increasing CQ-resistance [35] that would constitute a risk factor of emergence of AQ-resistance in Africa. However, high prevalence of SVMNT has been reported in P. falciparum from South America and Asia [36] and the presence of this haplotype was also reported in Tanzania [37] and in Angola [38]. AQ is structurally related to CQ with both belonging to the group of 4-aminoquinolines. There is a possibility that AQ could continue to contribute to selection for the mutant pfcrt-K76T parasites even after discontinuance of CQ usage [39].

The present study contributed to ongoing malaria surveillance providing baseline data for the biennial surveillance of P. falciparum molecular markers associated with resistance to anti-malarial drugs in the DRC. Regular studies with size of samples taking into account the regional variability and a broader exploration of the genome of P. falciparum could help to provide more information about the landscape of P. falciparum drug resistance in the DRC.