Resistance of Plasmodium falciparum to Sulfadoxine-Pyrimethamine ( Dhfr and Dhps ) and Artemisinin and Its Derivatives ( K 13): A Major Challenge for Malaria Elimination in West Africa

The spread of resistance to antimalarials is a major public health problem worldwide and especially in sub-Saharan Africa where the highest morbidity and mortality rates are found with a critical scarcity of data on resistance. The objec-tive of this review is to describe the mutations in the pfdhfr, pfdhps and k13 genes associated with resistance to artemisinin and Sulfadoxine-Pyrimethamine reported in West Africa during the decade 2007 to 2017 followed by a meta-analysis of their prevalence. A bibliographic search on the MEDLINE, PubMed, EMBASE and Sciences Direct databases made it possible to find 405 scientific papers relating to resistance to artemisinin and to Sulfadoxine-Pyrimethamine during the period 2007-2017. The analysis has concerned 217 resistant to artemisinin-based combination therapy in the West African region, increased surveillance is necessary to prevent the rapid occurrence of possible resistance, especially in the context of synonymous or non-K13 mutations correlated with a delay in parasitic clearance.


Introduction
In 2017, WHO estimated that 219 million people experience a malarial illness worldwide with 435,000 deaths, more than 90% of them in tropical Africa and 61% of children under five years of age [1]. About 41.1 million of morbidity and 18,400 deaths were recorded in West Africa [1]. This disease represents a serious threat to health systems in sub-Saharan Africa where morbidity and mortality from malaria are the highest and inadequate surveillance systems to better control its spread [2] [3] [4]. One of the major challenges in malaria elimination is the resistance of Plasmodium falciparum to antimalarials today. According to WHO recommendations, artemisinin and Sulfadoxine-pyrimethamine (SP) are currently the most used drugs as the first line of treatment against malaria in Africa [5]. But a decade ago, the first cases of parasites resistant to artemisinin and its derivatives were detected in western Cambodia and then spread to the area of Southeast Asia [6]. In the case of Sub-Saharan Africa, the data in the literature are controversial on the probable relationship between the presence of k13 mutants and resistance to artemisinin [4] [7]. On the other hand, cases of resistance to SP have already been demonstrated by several authors, while SP continues to be offered as an intermittent and preventive treatment against malaria [5] [8] [9] [10] [11]. In the current context of West Africa, the insufficiency of information and the divergence of the conclusions of most of the studies on the Plasmodium resistance genes to SP and to artemisinin contribute to disseminate uncertainties on the choice of certain molecules such as SP for intermittent preventive treatment (IPT) and the probable presence of mutations in the k13 gene associated with resistance to artemisinin [12]. To prevent resistance in this area, strict diagnosis of malaria infections prior to treatment and good compliance with antimalarial drugs accompany WHO recommendation of artemisinin-based combinations (ACT) for the management of simple malaria and the administration of 3 doses of SP in IPT in each pregnancy [3] [13] [14]. In the absence of a vaccine with sufficient and lasting efficacy, preserving the efficacy of antimalarials, in particular artemisinin and SP in IPT, therefore constitutes a major challenge for Sub-Saharan Africa and in particular for West African in malaria control.
A synthesis of work on the resistance of Plasmodium to these main antimala- rials is necessary to guide public health policies for the elimination of malaria in endemic areas of sub-Saharan Africa. To take stock of possible resistance to artemisinin and SP, this review will generally describe the situation of the resistance genes pfdhfr, pfdhps and k13 in West Africa during the decade 2007 to 2017. It will then be focused on a meta-analysis of the prevalence of mutations reported by the included studies in order to provide information that can facilitate decision-making on the effectiveness of SP and artemisinin.

Sulfadoxine-Pyrimethamine Mechanism of Action
Sulfadoxine (sulfonamide) is an antibiotic that inhibits the metabolism of folic acid (vitamin B9). Folic acid is essential for Plasmodium development [15]. Plasmodium synthesizes folic acid in 2 enzymatic steps using dihydropteroate synthetase (DHPS) then dihydrofolate reductase (DHFR). Blocking one of these pathways prevents the development of the parasite. Thus, sulfadoxine would inhibit the first synthetic enzyme, dihydropteroate synthetase (DHPS) and pyrimethamine would act on the second enzyme called dihydrofolate reductase (DHFR). SP has an erythrocyte and tissue schizonticidal effect, the action of which is prolonged by sulfadoxine. SP is most often recommended for intermittent and preventive treatment, especially in pregnant women [14] [16] [17].

Resistance to Sulfadoxine-Pyrimethamine
At the genetic level, resistance to SP is caused by mutations in the dhfr and dhps genes of Plasmodium falciparum. Mutations in the dhfr gene cause resistance to pyrimethamine; they are amino acid substitutions on codons S108N, N51I, C59R and I164L. For the dhps gene, the substitutions responsible for sulfadoxine resistance are located on codons S436A/F, A437G, K540E, A581G and A613T/S [18].

The Mechanism of Action of Artemisinin and Its Derivatives
Artemisinin or qinghaosu is a sesquiterpene lactone extracted from the leaves of a plant called Artemisia annua. Its biosynthesis is not yet fully understood [19].
The semi-synthetic derivatives of artemisinin are dihydroartemisinin (DHA), artesunate, artemether and arteether [20]. Artemisinin and its derivatives are pro-drugs that act on the schizonts of Plasmodium in erythrocytes. They cross the membrane of the red blood cells and then that of the parasites and accumulate in the digestive vacuoles of the parasite. Two main mechanisms of action are attributed to them. It would be the blocking of a SERCA (Sarco/Endoplasmic Reticulum Ca 2+ ATPase) or PfATPase enzyme which would allow the parasite to pump calcium for its development [19] [21]. The other mechanism of action results from the presence in the structure of artemisinin of an endoperoxide bridge playing a major role in the effectiveness of the molecule. The activation of the endoperoxide bridge during the endo-erythrocytic phase generates free radicals which alter the membrane of the parasite thus causing its death by oxidative stress [22] [23]. There are two ways of activating the endo-peroxide bridge. The mitochondria are said to be the seat of the first pathway which is caused by the electron transport chain, the consequence of which is a large production of Reactive Oxygen Species (ROS) [7]. The second way takes place in the digestive vacuoles thanks to the heme (Fe 2+ ) resulting from the catabolism of hemoglobin [24]. Artemisinin is toxic to chloroquine resistant strains. It acts mainly on rings and trophozoites in the growth phase. Its toxicity on the early stages of gametocytes gives it effectiveness in inhibiting the transmission of the parasite [19]. Artemisinin is however inactive on merozoites, pre-erythrocytic forms and other forms present in the parasite development cycle at the level of the malaria vector [19]. The World Health Organization issued guidelines in 2015 to recommend Artemisinin and its derivatives as first-line malaria treatment and two artemisinin derivatives can be used together. Children and adults with uncomplicated malaria in endemic area are strongly recommended to be treated with one of the following artemisinin-based combination therapies (ACT): artemether + lumefantrine, artesunate + amodiaquine, artesunate + mefloquine, dihydroartemisinin + piperaquine, artesunate + sulfadoxine pyrimethamine (SP).

Resistance to Artemisinin and Derivatives
Resistance to artemisinin and its derivatives results from certain mutations in the Kelch or k13 gene located on chromosome 13 of Plasmodium falciparum [25] [26] [27]. Any mutation in the Kelch 13 gene does not systematically confer resistance to artemisinin; two main criteria validate the mutations as being associated with artemisinin resistance. Resistance should be correlated with slow parasitic clearance in clinical studies and reduced sensitivity of the drug in vitro [28]. The assessment of drug sensitivity is based on the quantitative microscopic measurement of delayed parasite clearance following the first days of treatment with ACT or artemisinin monotherapy. This results in a longer parasite clearance half-life. The half-life parameter is measurable by the phenotype ring-stage survival assay or RSA which measures the parasite survival rate in the stage of young trophozoites at an exposure of 700 nM of dihydroartemisinin for 6 h [29]. WHO defines eight K13 mutants associated with the resistance of Plasmodium falciparum to artemisinin which are F446I, P553L, N458Y, R561H, M476I, C580Y, Y493H, R539T and I543T. The other mutants with one of the criteria described above are classified as associated candidates [28]. Resistance to an artemisinin derivative or ACT is considered resistance to artemisinin [30].

Statistical Analysis
The prevalence of the mutations was calculated with the Epi-info version 6 software and the RevMan 5.3 software was used for the meta-analysis. The Cochran's Q test was used to calculate the percentage of the total variance (I2) between the studies involved. "I2" reflects the heterogeneity between these studies. It is weak for the values of I2 ≤ 25%, moderate for 25% < I 2 ≤ 50% and strong for I 2 ≥ 75% [31]. In this comparison, two (2) meta-analyzes concerned the publications

Dhfr and Dhps Genes in West Africa
In

K13 Mutants in West Africa
In 2016 none of the previously described substitutions had been observed generally in sub-Saharan Africa apart from the P553L mutation which was observed at low frequencies in Kenya (0.53%) and in Malawi (0.59%) [

Meta-Analysis of SP Resistance Data in Plasmodium falciparum
A first meta-analysis of the data showed strong heterogeneity (I 2 > 90%) between the studies compared (Tables 1-4

Dhfr and Dhps Genes in West Africa
The use of SP in West Africa for the intermittent and preventive treatment of malaria has not so far been associated with a loss of birth weight and a drop in  26 -48.42) the level of maternal hemoglobin [41]. The distribution of mutations at codons 59, 540 and triple/quadruple/quintuple mutations of the pfdhfr and pfdhps genes would be highly predictive of treatment failures in SP [42]. Most of these SP resistance markers present at the majority of sites in West Africa could seriously compromise the effectiveness of intermittent preventive treatment for years to come [10] [43]. This situation would call for new approaches and new strategies with regard to the efficient use of SP in West Africa and in general in sub-Saharan Africa.

Meta-Analysis of SP Resistance Data in Plasmodium falciparum
The first meta-analysis revealed a large disparity (I 2 > 90%) between the studies compared, which would not make the result obtained credible. A second comparison in the absence of certain studies made it possible to obtain better results with low or moderate heterogeneity. Analysis of the bias between the studies considered shows that there is a likely influence of the type of study and prevalence. This bias was minimized during the second comparison. The prevalence of each Dhps (A437G) or Dhfr mutant (N51I, C59R, S108N) is relatively high, unlike that of the triple Dhfr mutation. These data would suggest that SP could still be recommended with caution in the intermittent preventive treatment against malaria in the West African Region. However, the residual risk of increasing malaria could be high in the coming years with the emergence of double, triple, quadruple and quintuple mutations [44].

Conclusion
Despite the increasing prevalence of dhfr and dhps mutants in the West African region, SP is still recommended for prevention against malaria in this area. In addition, the emergence of triple, quadruple or quintuple Dhfr/Dhps mutation could in the near future dangerously jeopardize the use of SP for intermittent preventive treatment in West Africa. However, artemisinin, its derivatives and artemisinin-based combinations (ACT) are said to be still effective in West Africa at this time. The challenge of protecting the effectiveness of ACT for this region, is to maintain a high wakefulness level in the monitoring to prevent the rapid onset of possible resistance to artemisinin.