Background
During the past 20 years, many strains of
Plasmodium falciparum have become resistant to chloroquine and other anti-malarial drugs [
1]. One strategy for reducing malaria prevalence is the use of drugs in combination. Drug combinations help prevent the development of resistance to each component drug and reduce the overall transmission of malaria [
2]. In response to increasing chloroquine resistance, Senegal in 2004 switched to sulphadoxine-pyrimethamine with amodiaquine as the first-line therapy. In 2006, artemether-lumefantrine and artesunate-amodiaquine were the forms of artemisinin-based combination therapy (ACT) recommended by the WHO as the first-line anti-malarial regimen for managing uncomplicated malaria. Since 2006, more than 1.5 million treatments have been administered in Senegal [
3]. During 2009, 184,170 doses of ACT were dispensed in Senegal [
4].
Dakar, the capital city of Senegal, has an urban population of approximately 1.1 million and a suburban population of 2.3 million; the city covers the majority of the Cap-Vert Peninsula. Malaria is transmitted in Dakar and its surrounding suburbs, with spatial heterogeneity of the human biting rate, which ranged from 0.1 to 250 bites per person per night during the rainy season from 2007 to 2010 [
5]. Intermittent preventive treatment (IPT) with anti-malarial drugs given to all children and pregnant women once per month during the transmission season can provide a high degree of protection against malaria. Seasonal IPT with sulphadoxine-pyrimethamine and one dose of artesunate resulted in a 90% reduction in the incidence of clinical malaria in Senegal [
6]. The combination of sulphadoxine-pyrimethamine and amodiaquine was more effective than the combination of sulphadoxine-pyrimethamine and artesunate or the combination of amodiaquine and artesunate in preventing malaria [
7]. During IPT with sulphadoxine-pyrimethamine and piperaquine, only 3.4% of the treated children developed malaria [
8].
Since the introduction of ACT and IPT trials in Senegal, there have been very few reports on the level of resistance of
P. falciparum to anti-malarial drugs. To determine whether parasite susceptibility has been affected by the new anti-malarial policies, a study of molecular markers was conducted with local isolates obtained from the military hospital of Dakar (Hôpital Principal de Dakar). The prevalence of genetic polymorphisms in genes associated with anti-malarial drug resistance was evaluated. The genes interest included
P. falciparum chloroquine resistance transporter (
Pfcrt) for chloroquine [
9],
P. falciparum dihydrofolate reductase (
Pfdhfr) for pyrimethamine [
10],
P. falciparum dihydropteroate synthase (
Pfdhps) for sulphadoxine [
11] and
P. falciparum multidrug resistance 1 (
Pfmdr1) for mefloquine resistance [
12] and potentially for quinoline resistance [
13,
14].
The
Pfcrt gene was firstly identified in 2000 [
9]. So far at least 20 mutation points were described [
9,
15,
16], but only one is the reference mutation, the marker of chloroquine resistant phenotype: K76 that becomes T76 when mutated. This mutation is often associated with other mutations in the
Pfcrt gene (Cys72Ser, Met74Ile, Asn75Glu, Ala220Ser, Gln271Glu, Asn326Ser, Ile356Thr, Arg371Ile). The role of these mutations is not yet defined. The odds ratio (OR) for failure associated with K76T mutation was 2.1 (95% confidence interval: 1.5-3.0, meta-analysis of 13 studies) for a 14-day follow-up and 7.2 (95%CI: 4.5-11.5, meta-analysis of 12 studies) for a 28-day follow-up [
17]. However, the existence of chloroquine-susceptible strains associated with K76T mutation suggests that other genes could be involved in resistance to chloroquine.
The Ser108Asn mutation on the
Pfdhfr gene is associated with resistance to anti-folate drugs [
18]. The OR for sulphadoxine-pyrimethamine failure associated with Ser108Asn was 3.5 (95%CI: 1.9-6.3, meta-analysis of 10 studies) for a 28-day follow-up [
17]. The additional mutations Asn51Ile, Cys59Arg or Ile164Leu increase the level of in vitro resistance to antifolate drugs and sulphadoxine-pyrimethamine. The OR for codon 51 and 59 single mutants were 1.7 (95%CI: 1.0-3.0) and 1.9 (95%CI: 1.4-2.6), respectively [
17]. The triple mutation (51+59+108) increases the risk of
in vivo resistance to sulphadoxine-pyrimethamine by 4.3 (95%CI: 3.0-6.3, meta-analysis of 22 28-day studies) [
17].
Sulphones (dapsone) and sulphonamides (sulphadoxine) are inhibitors of
P. falciparum DHPS [
19]. The mutations Ser436Ala, Ser436Phe, Ala437Gly and Lys540Glu are involved in resistance to sulphadoxine [
11]. The single mutation Ala437Gly and the double mutation Ala437Gly+Lys540Glu increase the risk of
in vivo resistance to sulphadoxine-pyrimethamine by 1.5 (95%CI: 1.0-2.4, meta-analysis of 12 studies) and 3.9 (95%CI: 2.6-5.8, meta-analysis of 10 studies), respectively [
17].
The quintuple mutant of
Pfdhfr (codons 51+59+108) plus
Pfdhps (codons 437+540) increases the risk of
in vivo resistance to sulphadoxine-pyrimethamine by 5.2 (95%CI: 3.2-8.8, meta-analysis of 3 studies) [
17].
Pfmdr1, which encodes a 162 kDa protein named
P. falciparum homologue of the P-glycoprotein (Pgh1), is located on chromosome 5. Field work has shown that the predictive value for chloroquine resistance and point mutations in the
Pfmdr1 sequence resulting in amino acid changes varies depending on the geographic area [
20,
21]. Five point mutations have been described: N86Y, Y184F, S1034C, N1042D and D1246Y. Point mutations, most notably N86Y, have been associated with a decrease in the chloroquine susceptibility [
22]. However, in some of these epidemiological studies, the number of chloroquine-susceptible samples is too limited to provide statistically meaningful analysis [
21,
23]. Using precautions, no or only weak relationships are established in
P. falciparum between chloroquine resistance and mutations in
Pfmdr1[
24]. However, the risk of therapeutic failure with chloroquine is greater for patients harbouring the N86Y mutation with an OR of 2.2 (95%CI: 1.6-3.1) with a 14-day follow-up and 1.8 (95%CI: 1.3-2.4) with a 28-day follow-up [
17]. The combination of
Pfmdr1 N86Y and
Pfcrt K76T increases the risk of
in vivo resistance to chloroquine by 3.9 (95%CI: 2.6-5.8, meta-analysis of 5 studies) [
17].
In addition, the risk of therapeutic failure with amodiaquine is greater for patients harbouring the N86Y mutation with an OR of 5.4 (95%CI: 2.6-11.2, meta-analysis of six studies) [
17]. This mutation increases the risk of failure with amodiaquine plus sulphadoxine-pyrimethamine by 7.9 [
25].
It has been shown through heterologous expression that
Pfmdr1 mutations at codons 1034 and 1042 abolish or reduce the level of resistance to mefloquine [
26]. Moreover, transfections with a wild-type
Pfmdr1 allele at codons 1034, 1042 and 1246 confer mefloquine resistance to susceptible parasites [
27]. However, mutations at codons 1034, 1042 and 1246 in
P. falciparum Pfmdr1 isolates are not sufficient to explain variations in mefloquine susceptibility [
28]. Analyses of
P. falciparum isolates showed an association between mutation at the codon 86 and an increase in susceptibility to mefloquine, halofantrine or artemisinin derivatives [
29‐
31].
Amplification and overexpression of
Pfmdr1 has been associated with mefloquine resistance and halofantrine decreased susceptibility in
P. falciparum[
32,
33] Recently, Price
et al. showed that amplification of
Pfmdr1 is the main cause of resistance to mefloquine in
P. falciparum[
12]; the
Pfmdr1 copy number could be used as a molecular marker to monitor mefloquine drug resistance in areas of emerging resistance [
29]. The OR for mefloquine failure in monotherapy associated with
Pfmdr1 amplification is 8.6 (95%CI: 3.3-22.9) at day 28 [
17]. Increased copy number from 1 to 2 is associated with a significant high risk of clinical failures with mefloquine-artesunate (OR = 2.6) [
17,
34] and artemether-lumefantrine [
17]. Increase of
Pfmdr1 copy is associated with
in vitro reduced susceptibility to artemisinin derivatives [
35‐
37]. However, increase of
Pfmdr1 copy seems to be not associated with
in vivo prolonged clearance time [
38,
39].
Discussion
In response to increasing chloroquine resistance, Senegal in 2004 switched to sulphadoxine-pyrimethamine with amodiaquine as the first-line therapy. In 2006, artemether-lumefantrine and artesunate-amodiaquine were the forms of artemisinin-based combination therapy (ACT) recommended by the WHO as the first-line anti-malarial regimen for managing uncomplicated malaria.
Mutations in
Pfcrt have been shown to be correlated with chloroquine resistance in different parts of the world [
43]. The prevalence of the
Pfcrt 76T mutation decreased since 2004 in Dakar. In 2000–2001 in Guediawaye, a suburb of Dakar, a prevalence of 92% of 76T was observed in pregnant women with malaria [
44]. In Pikine, another suburb of Dakar, the prevalence of 76T was 79% in 2000 [
45], 63.9% in 2001 [
46] and 59.5% in 2004 [
47]. In 2002, the prevalences of
in vitro resistance to chloroquine and of
Pfcrt 76T mutation were 52% and 65%, respectively, in patients hospitalized for malaria at the Hôpital Principal de Dakar [
48]. In 2001–2002, the prevalence of the
pfcrt 76T mutation was 75.8% in pregnant women taking chloroquine prophylaxis in Thiadiaye (84 km southeast of Dakar) [
49]. In Dielmo (280 km southeast of Dakar), the
in vitro resistance to chloroquine regularly increased from 32% in 1995 to 55% in 1999 [
50‐
53].
In this study, the
Pfcrt 76T mutation was identified in 37.2% of the patients recruited from October 2009 to January 2010 in the Hôpital Principal de Dakar. These data are consistent with previous works on molecular resistance and on
in vitro or
ex vivo susceptibility in Dakar in 2009 (22% of isolates exhibiting chloroquine resistance) [
54] and in Thies in 2007 (23% of isolates exhibiting chloroquine resistance) [
55].
This decrease in chloroquine resistance parallels the withdrawal of chloroquine treatment and the introduction of ACT in 2002 in Senegal. However, in 2003, chloroquine was still being administered to patients. The prevalence of chloroquine in the urine ranged from 14.5% to 47.5% in two- to nine-year-old children from northern Senegal and from 9.0% to 21.4% in children from southern Senegal [
56]. In 2006, Senegal reported 10.6% chloroquine use and 9.7% ACT use [
57]. Since 2006, more than 1.5 million ACT treatments have been administered in Senegal [
3], and 184,170 doses of ACT were dispensed in 2009 [
4]. A reduction in chloroquine resistance was also reported in Malawi after the withdrawal of chloroquine treatment [
58]. This observation prompted an
in vivo chloroquine study in Malawi five years later, in which chloroquine was found to be 99% effective [
59]. The rapid dissemination of chloroquine resistance in Dielmo, despite strictly controlled anti-malarial drug use, argues against the re-introduction of chloroquine at least in mono-therapy in places where the resistant allele has dropped to very low levels following the discontinuation of chloroquine treatment [
60]. It took 407 chloroquine treatments (1.6 treatment courses/person/year) in the community to raise the prevalence of the
Pfcrt 76T mutation from an 8-9% during the first year of re-introduction of chloroquine (1993–1994) to 46% in 1995. Increased selective pressure (2752 treatments during the period 1995–1999) did not increase the prevalence of
Pfcrt 76T or increased the
in vitro resistance to chloroquine, but this increased selective pressure increased the incidence of clinical malaria for patients within seven days of chloroquine treatment from 2.6% in 1995 to 13% in 1999.
The
Pfdhfr 108N mutation has been shown to be correlated with
in vitro and
in vivo resistance to pyrimethamine [
10,
17]. The OR for sulphadoxine-pyrimethamine failure associated with Ser108Asn was 3.5 (95%CI: 1.9-6.3, meta-analysis of 10 studies) for a 28-day follow-up [
17]. The additional mutations Asn51Ile, Cys59Arg or Ile164Leu increase the level of in vitro resistance to anti-folate drugs and sulphadoxine-pyrimethamine. The OR for codon 51 and 59 single mutants were 1.7 (95%CI: 1.0-3.0) and 1.9 (95%CI: 1.4-2.6), respectively [
17]. In 2009, the prevalence of
Pfdhfr 108N was 82.4% in patients with malaria who were treated at the Hôpital Principal de Dakar. The triple mutation (51+59+108) increases the risk of
in vivo resistance to sulphadoxine-pyrimethamine by 4.3 (95%CI: 3.0-6.3, meta-analysis of 22 28-day studies). Isolates carrying a combination of three mutations (108N, 51I and 59R) associated with high-level pyrimethamine resistance represented 75.3%. In 2002, in the same hospital, the prevalence of
Pfdhfr 108N was 65%, and triple mutants were identified in 50% of the isolates [
48]. In 2003, the prevalence of mutations in
Pfdhfr codon 108 was 78% in Pikine, and the prevalence of the triple mutant was 61% [
61]. In 2007 in Keur Soce, a rural area, triple mutant was identified in 67% of patients treated with sulphadoxine-pyrimethamine combined with amodiaquine [
8].
The
Pfdhps 437G mutation has been shown to be correlated with
in vitro and
in vivo resistance to sulphadoxine [
11,
17]. The single mutation Ala437Gly and the double mutation Ala437Gly+Lys540Glu increase the risk of
in vivo resistance to sulphadoxine-pyrimethamine by 1.5 (95%CI: 1.0-2.4, meta-analysis of 12 studies) and 3.9 (95%CI: 2.6-5.8, meta-analysis of 10 studies), respectively [
17]. In 2009, the prevalence of the
Pfdhps 437G mutation was 40.4% in patients with malaria who were treated at the Hôpital Principal de Dakar. However, there was no isolate carrying the double mutation (437G and 540E) that is associated with high-level sulphadoxine resistance. The mutation of codon 613 (A613S) (1.8%) was very rare in Africa. In 2002, in the same hospital, only 20% of isolates harboured the
Pfdhps 437G mutation [
48]. In 2003, the mutation rate in
Pfdhps codon 437G was 40% in Pikine [
61]. Several studies from 2006 to 2008 in Senegal showed that the prevalence of
Pfdhps 437G significantly increased after intermittent preventive treatment of infants with sulphadoxine-pyrimethamine [
8,
62]. Given the prevalences of the triple and quadruple mutants in the population of Dakar (75.3%
Pfdhfr 108N, 51I and 59R triple mutant and 36.5% quadruple mutant
Pfdhfr 108N, 51I and 59R and
Pfdhps 437G), the use of sulphadoxine-pyrimethamine as an intermittent preventive treatment must be monitored. Encouragingly, no quintuple mutant (
Pfdhfr 108N, 51I and 59R and
Pfdhps 437G and 540E), which is associated with high-level sulphadoxine-pyrimethamine resistance, has been identified to date. However, the single use of sulphadoxine-pyrimethamine as seasonal IPT must be inadvisable: sulphadoxine-pyrimethamine must be associated with amodiaquine, artesunate or piperaquine for instance [
7,
8].
Only one isolate (0.6%) had two copies of
Pfmdr1. In Asia, amplification of
Pfmdr1 is associated with mefloquine resistance
in vitro and
in vivo[
12,
29,
63]. The role of increased copy number in mefloquine resistance in Africa remains controversial. Isolates with a duplicated
Pfmdr1 gene circulate in West Africa but are relatively rare [
64]. Only one published clinical failure for mefloquine in West Africa was found to be associated with
in vitro resistance and amplification of
Pfmdr1[
65]. In addition, amodiaquine resistance is not related to the amplification of
Pfmdr1[
66]. The role of the amplification of
Pfmdr1 in resistance to artemether-lumefantrine in Africa is still debated. It seems that no
Pfmdr1 gene amplification was associated with artemether-lumefantrine failures in Africa [
67], whereas a copy number ≥4 is associated with reduced
in vitro susceptibility to lumefantrine [
68]. In 2009, in Dakar, only 1% of the isolates presented reduced
in vitro susceptibility to lumefantrine [
44], and this prevalence did not increase in Senegal after the introduction of ACT. In 1996, 6% of isolates from Dielmo were resistant
in vitro to lumefantrine [
69]. In recent years, the efficacy of artemether-lumefantrine in several trials in Senegal has ranged from 96 to 100% [
70‐
72].
The prevalences of the
Pfmdr1 mutations 86Y and 184F were 16.6% and 67.6%, respectively. No isolate carried a mutation in codons 1034, 1042 or 1246. In 2000 and 2001, prevalences of 31% and 30.6 were observed for
Pfmdr1 86Y in Pikine [
45,
46]. The role of polymorphism in
Pfmdr1 is still debated. Point mutations in
Pfmdr1, most notably at codon 86, have been found to be associated with decreased chloroquine susceptibility [
73]. Nevertheless, this association is not a consistent finding [
24]. The
Pfmdr1 86Y mutation also has been found to be associated with increased susceptibility to mefloquine or artemisinin [
29‐
31]. This association, too, is not a consistent finding [
63]. In addition, no clear association between the
Pfmdr1 184F mutation and mefloquine failure has been established, although this allele is widespread in Cambodia [
74]. The
Pfmdr1 86Y mutation has also been shown to be associated with
in vivo resistance to amodiaquine in recrudescence after monotherapy with amodiaquine [
75] or after combination therapy with artesunate-amodiaquine [
76]. The
Pfmdr1 1246Y mutation has also been found to be associated with
in vitro resistance to amodiaquine [
77] and with recrudescent infection after treatment with amodiaquine or amodiaquine-artesunate [
76,
78]. In a meta-analysis, the
Pfmdr1 86Y mutation was demonstrated to be associated with amodiaquine failure, with an odds ratio of 5.4 [
17]. Based on this hypothesis, the 16.6% prevalence of
Pfmdr1 of 86Y predicts that 16.6% of isolates would be resistant to amodiaquine in 2009 in Senegal. In 2009, in Dakar, only 6% of isolates showed
in vitro reduced susceptibility to monodesethylamodiaquine, the active metabolite of amodiaquine [
54]. The resistance to amodiaquine has remained low even after the introduction of artesunate-amodiaquine in 2006 in Senegal relative to the resistance prevalences of Dielmo in 1996 and 1999 (0%) [
50,
79] and Mlomp in Casamance, south-western Senegal, in 2004 (5%) [
80]. The artesunate-amodiaquine–associated cure rates were >99.3% in Mlomp and Keur-Socé when administered either as a single daily dose or two daily doses [
81]. The fixed-dose combination of artesunate-amodiaquine (ASAQ) exhibits a cure rate >98.5% [
82]. The cure rates were 100% in the populations experiencing a second or third episode of uncomplicated malaria following treatment with ASAQ [
69]. However, ACT efficacy and resistance must be monitored because the first clinical failures, or at least extended parasite clearance times, have been described in Cambodia [
38,
83]. In this context, it is important to implement
in vitro and
in vivo surveillance programmes, such as those championed by the Worldwide Antimalarial Resistance Network [
84,
85].
Since 2004, the prevalence of chloroquine resistance has decreased, but the data argue against the re-introduction of chloroquine at least in mono-therapy in places where the resistant allele has dropped to very low levels following discontinuation of chloroquine treatment. The prevalence of isolates resistant to pyrimethamine is high (82.4%), with 75.3% of parasites exhibiting high-level pyrimethamine resistance. The prevalence of isolates resistant to sulphadoxine was 40.2%. However, no quintuple mutant (Pfdhfr 108N, 51I and 59R and Pfdhps 437G and 540E), which is associated with high-level sulphadoxine-pyrimethamine resistance, has been identified to date. The resistance to amodiaquine remains moderate. Intensive surveillance of P. falciparum susceptibility to anti-malarial drugs must be conducted regularly in Senegal. However, maximizing the efficacy and longevity of ACT as a tool to control malaria will critically depend on pursuing intensive research into identifying in vitro markers as well as implementing in vitro and in vivo surveillance programs. In this context, there is a need to identify molecular markers that predict ACT resistance which can provide an active surveillance method to monitor temporal trends in parasite susceptibility.
Authors’ contributions
NW, AP, EB and SB carried out the molecular genetic studies. BF, SD, SD, TD, BD, KBF, PSM and FF carried out diagnostic tests, monitored the patients, collected clinical and epidemiological data and drafted the manuscript. CR, RB, BW and BP conceived and coordinated the study. SB, CR and BP analysed the data. NW, AP, SB and BP drafted the manuscript. All authors read and approved the final manuscript.