Discussion
This report describes the evaluation of the ex vivo susceptibility of P. falciparum isolates, taken from patients in Dakar, to ten standard or potential anti-malarial drugs. The patients, recruited at the Hôpital Principal de Dakar from November 2013 to January 2014, said that they did not leave Dakar and its surrounding suburbs during the month preceding their malaria attack.
One limitation of this study was the low number of recruited patients (24) during those two months, due to the diminution of malaria prevalence in Senegal. The malaria prevalence in public health facilities decreased from 17.9% in 2007 to 2.6% in 2008 in Dakar [
11]. In Dielmo, a village located at 280 km southeast of Dakar and approximately 15 km north of The Gambia border, the prevalence of malaria decreased from 87.2 to 0.3% in children and 58.3 to 0.3% from 1990 to 2012 [
12].
The prevalence of isolates with reduced susceptibility to MQ remained high (55.6%) in Dakar, but was relatively stable compared with the previous year (55 to 62%) [
3,
10]. The level of
in vitro MQ resistance increased since previous studies conducted in Dakar. In Dakar, the per cent of isolates with decreased susceptibility was 17% in 2001 [
13] and 13% in 2002 [
7]. MQ prophylaxis failure has been previously described in Senegal [
14], and MQ is one of the three anti-malarial drugs recommended for travellers as an anti-malarial prophylaxis in Senegal. Clinical trials are in progress to evaluate the efficacy of MQ for intermittent preventive treatment of infants and pregnant women, whereas MQ is still used for the treatment of uncomplicated malaria in infants in Dakar. Nevertheless, MQ has been employed relatively infrequently in Africa compared to Asia. The combination of artesunate-mefloquine, which is administered to patients in Asia, is not yet used in Senegal. However, scientific data are not available for MQ monotherapy, and very little data are available on the
in vitro decreased susceptibility to MQ and its clinical implications in Africa. It is important to monitor the evolution of
P. falciparum susceptibility to MQ, to archive suspicious isolates and to correlate clinical outcomes with pharmacokinetic and phenotypic responses and with molecular markers.
As far back as 1988,
in vitro P. falciparum resistance to CQ was reported in Dakar, and reports of resistance in other regions of the country followed shortly thereafter [
15]. From 1991 to 1995, parasitological failures were observed in 21% of patients in Pikine [
16]. The prevalence of
in vitro CQ resistance then decreased in from 52% in 2002 [
7] to approximately 20-25% in Dakar in 2009–2011 [
3,
10]. In 2013–2014, the prevalence of
in vitro resistance to CQ in Dakar increased again to 50%. A limitation of these results is the very small number of studied samples. However, this phenomenon was already described in Thies and Pikine. Parasites also became less susceptible to CQ from 2008 (median IC
50 = 30.7 nM) to 2011 (median IC
50 = 76.1 nM) in Thies [
4]. In Pikine, after a decrease of the prevalence of the
pfcrt 76 T mutation, involved in CQ resistance, from 64-79% before CQ withdrawal (2000 to 2003) [
17-
19] to 47-60% [
20,
21] when amodiaquine plus pyrimethamine-sulphadoxine was the first-line treatment (2004–2005), this prevalence has increased slightly to 59% since ACT has been implemented (2006 to 2009) [
19]. It is important to monitor the evolution of
P. falciparum susceptibility to CQ.
The decrease in CQ resistance parallels the withdrawal of CQ treatment and the introduction of ACT in 2002 in Senegal. However, in 2006, CQ was still being administered to patients. In Dakar in 2006, CQ represented 5.1% of the anti-malarial drugs used in children [
22] and 3.5% in 2009 [
23]. The rapid dissemination of CQ resistance in Dielmo, despite strictly controlled anti-malarial drug use, argues against the re-introduction of CQ, at least in monotherapy, in places where the resistance allele has dropped to very low levels following the discontinuation of CQ treatment [
24]. Despite the re-aquisition of CQ susceptibility, any re-introduction would likely result in a rapid re-emergence of resistant strains. Additionally, the increase of CQ resistance in the hypothetical absence of CQ pressure leads to an avoidance of re-introducing CQ in Senegal. There is an urgent need to evaluate the presence and use of CQ in Dakar and to evaluate the capacity of drug pressure on CQ resistance of the different partners combined with artemisinin derivatives in ACT.
The prevalence of isolates with
in vitro-reduced susceptibility to MDAQ remains low and stable in 2013 (5.6
versus 6% in 2009 and 11.8% in 2010) [
3,
10]. The resistance to amodiaquine has remained low even after the introduction of artesunate-amodiaquine in 2006 in Senegal. A study in Dakar and Mlomp from 1996 to 1998 demonstrated that monotherapy with amodiaquine remained effective for treating uncomplicated malaria in areas where CQ resistance was prevalent [
25]. In 2011–2012, the efficacy of ASAQ was evaluated at 99.3% [
26]. However, ACT efficacy and resistance must be monitored because clinical failures, or at least extended parasite clearance times, have been described in Southeast Asia [
27-
30]. In this context, it is important to implement
in vitro and
in vivo surveillance programmes.
No isolate exhibited reduced
in vitro susceptibility to DHA or to AS. This result is consistent with previous studies that did not show any parasites resistant to AS [
7,
31,
32]. However, high IC
50 values can be found for artemisinin, with an IC
50 > 30 nM in Dakar [
33] and AS with an IC
50 > 45 nM [
13]. The median IC
50 values increased from 2008 to 2011 (3.2 to 10.1 nM) with a highest IC
50 value of 73.1 nM in Thies [
4]. In the present study, IC
50 ranged from 0.1 to 5.08 nM for DHA and 0.12 to 8.53 nM for AS. However, the standard
in vitro test was not adapted to follow resistance to artemisinin derivatives. The clinical resistance to artemisinin correlated with
in vitro resistance, manifested by an increase in the ring-stage survival rate after contact with artemisinin (ring survival test) [
34,
35]. In addition, mutations in the
P. falciparum K13 gene (PF3D7_1343700) that encodes the kelch propeller domain were associated with
in vitro resistance to artemisinin and with delayed clearance after artemisinin treatment in southern Asia [
30,
36,
37]. It will be a priority to introduce this new
in vitro test in Senegal.
The other ACT first-line treatment for uncomplicated
P. falciparum malaria in Senegal is the combination of artemether-lumefantrine. No isolate presented reduced susceptibility to LMF, and this prevalence was remains under 3% in Dakar since the introduction of ACT [
3,
10]. In 2011–2012, the efficacy of artemether-lumefantrine was evaluated at 100% in Senegal [
26].
A new ACT second-line treatment for uncomplicated
P. falciparum malaria in Senegal is the combination of dihydroartemisinin-piperaquine (DP). DP (Artekin®, Duo-Cotecxin®, Eurartesim®) is administered as single daily dose for three days. It has been demonstrated to be well tolerated and highly effective for the treatment of uncomplicated
P. falciparum malaria in Africa [
38-
40]. In 2011–2012, the efficacy of DP was evaluated at 100% in Senegal [
26]. The PPQ IC
50 values (geometric mean IC
50 = 32.2 nM) observed in Dakar in 2013 were slightly lower than those found in other
ex vivo studies in Africa (geometric mean IC
50 = 81.3 nM and 66.8 nM) [
41,
42].
The pyronaridine-artesunate combination (Pyramax®) is one of the most recent ACT combinations and is currently under development by the not-for-profit organisation Medicines for Malaria Venture (Geneva, Switzerland) and the pharmaceutical company Shin Poong Pharmaceuticals (Seoul, Republic of Korea) for the treatment of uncomplicated
P. falciparum malaria and for the blood stages of
Plasmodium vivax malaria. The efficacy of PND-artesunate was not inferior to that of artemether-lumefantrine in the treatment of uncomplicated
P. falciparum malaria in Africa [
43]. The PND IC
50 values (geometric mean IC
50 = 5.8 nM) observed in Dakar in 2013 were comparable to those obtained in Dielmo in 1996 and 1997 (3.8 nM and 4.52 nM) [
44,
45].
In 2013, 7% of isolates showed low reduced susceptibility to QN, which is in accordance with previous studies in Dakar [
3,
7,
10,
13]. Even in areas where QN efficacy remains good, such as sub-Saharan Africa, the susceptibility of individual
P. falciparum isolates to QN has varied widely. The IC
50s for isolates collected in Dakar were 6 to 1,291 nM in 2009 [
10] and 5 to 1,195 nM in 2010 [
3]. The wide range in QN susceptibility and recent evidence for QN treatment failure observed across Africa [
46,
47] or in Senegal in a patient who spent two months in Dielmo in 2007 [
48] suggest that the evolution of parasites with reduced susceptibility may contribute to QN decreased efficacy. However, the 24 patients in this study were successfully treated with QN.
Proveblue (PVB), which is a methylene blue preparation that complies with the European Pharmacopoeia and contains limited organic impurities and heavy metals of recognized toxicity, has previously been demonstrated to possess
in vitro anti-malarial activity against 23
P. falciparum strains that were resistant to various anti-malarial drugs [
49]. PVB exhibited noticeable synergistic effects in combination with MQ and QN and high synergistic effects associated with DHA [
50]. Treatment with 1 to 10 mg/kg of weight of PVB for five days significantly reduced or prevented cerebral malaria in mice [
51-
53]. The IC
50 for PVB ranged from 0.88 nM to 40.2 nM with a mean of 5.3 nM. These data show that PVB is active
in vitro, in line with previous studies with methylene blue with organic as well as inorganic impurities in parasites from Nigeria, Kenya and Thailande [
54-
56]. Another advantage of the use of PVB is that methylene blue has gametocytocidal properties and can reduce the transmission of
P. falciparum [
57,
58].
Limitations must be taken into account such as the very small number of samples which are not representative of susceptibility in Senegal but only from a facility in Dakar, the Hôpital Principal de Dakar, which certainly selects most severe malaria than in neighborhood clinics.
The introduction of ACT in 2002 in Senegal did not induce a decrease in P. falciparum susceptibility to individual drug components, such as DHA, AS, MDAQ, and LMF. The prevalence of P. falciparum isolates with reduced drug susceptibility to MQ remains high and stable in Dakar. Since 2004, the prevalence of CQ resistance has decreased, but then rebounded in 2013 in Dakar. PND, PPQ and PVB showed high in vitro activity against P. falciparum parasites from Dakar. 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 ex vivo and in vivo surveillance programmes.
Authors’ contributions
BF, YD and BP carried out the in vitro testing of drug susceptibility. CC, MF, PD, and BD supervised, carried out and coordinated the field collection of isolates from patients. BW and BP conceived and coordinated the study. BP analysed the data. BF and BP drafted the manuscript. All authors read and approved the final manuscript.