Skip to main content

Plasmodium falciparum susceptibility to standard and potential anti-malarial drugs in Dakar, Senegal, during the 2013–2014 malaria season

Abstract

Background

In 2006, the Senegalese National Malaria Control Programme recommended artemisinin-based combination therapy (ACT) as the first-line treatment for uncomplicated malaria. Since the introduction of ACT, there have been very few reports on the level of Plasmodium falciparum resistance to anti-malarial drugs. An ex vivo susceptibility study was conducted on local isolates obtained from the Hôpital Principal de Dakar (Dakar, Senegal) from November 2013 to January 2014.

Methods

Eighteen P. falciparum isolates were sussessfully assessed for ex vivo susceptibility to chloroquine (CQ), quinine (QN), monodesethylamodiaquine (MDAQ), the active metabolite of amodiaquine, mefloquine (MQ), lumefantrine (LMF), artesunate (AS), dihydroartemisinin (DHA), the active metabolite of artemisinin derivatives, pyronaridine (PND), piperaquine (PPQ), and, Proveblue (PVB), a methylene blue preparation, using the HRP2-based ELISA test.

Results

The prevalence of isolates with reduced susceptibility was 55.6% for MQ, 50% for CQ, 5.6% for QN and MDAQ, and 0% for DHA, AS and LMF. The mean IC50 for PND, PPQ and PVB were 5.8 nM, 32.2 nM and 5.3 nM, respectively.

Conclusions

The prevalence of isolates with a reduced susceptibility to MQ remains high and stable in Dakar. Since 2004, the prevalence of CQ resistance decreased, but rebounded in 2013 in Dakar. PND, PPQ and PVB showed high in vitro activity in P. falciparum parasites from Dakar.

Background

In response to increasing chloroquine resistance, Senegal switched in 2004 to sulphadoxine-pyrimethamine with amodiaquine as the first-line therapy for malaria. In 2006, the Senegalese National Malaria Control Programme recommended artemisinin-based combination therapy (ACT) as the first-line treatment for uncomplicated malaria. The combined sulphadoxine-pyrimethamine and amodiaquine treatment was changed to artemether-lumefantrine or artesunate-amodiaquine. Since 2006, more than 1.5 million ACT-based treatments have been administered in Senegal [1]. In 2006, the Senegalese National Malaria Control Programme also recommended testing all suspected cases of malaria with the Plasmodium falciparum histidine-rich protein 2 (PfHRP2)-based rapid diagnostic test (RDT). Since this time, ACT use has been restricted to confirmed malaria cases to reduce drug resistance. In 2009, 184,170 doses of ACT were dispensed at public health facilities in Senegal [2].

Since the introduction of ACT, there have been very few reports on the level of P. falciparum resistance to anti-malarial drugs. The last ex vivo susceptibility study was conducted in 2010 in Dakar (Médina district) [3] and in 2011 in Thies [4]. To determine whether parasite susceptibility has been affected by the new anti-malarial policies, an ex vivo susceptibility study was conducted on local isolates from Dakar obtained from the Hôpital Principal de Dakar between November 2013 and January 2014. The malaria isolates were assessed for susceptibility to standard drugs such as chloroquine (CQ), quinine (QN), monodesethylamodiaquine (MDAQ), the active metabolite of amodiaquine, mefloquine (MQ), lumefantrine (LMF), artesunate (AS), dihydroartemisinin (DHA), the active metabolite of artemisinin derivatives, and new anti-malarial drugs, such as pyronaridine (PND), piperaquine (PPQ) and Proveblue (PVB), a methylene blue preparation that complies with the European Pharmacopoeia and contains limited organic impurities and heavy metals of recognised toxicity.

Methods

Plasmodium falciparum isolates

In total, 24 patients (seven females and 17 males) with malaria were recruited from 19 November 2013 to 7 January 2014 at the Hôpital Principal de Dakar. Venous blood samples were collected in Vacutainer® ACD tubes (Becton Dickinson, Rutherford, NJ, USA) prior to patient treatment. Parasitaemia ranged from 0.001 to 0.33% in the male group and from 0.001 to 3.3% in the female group. Of the 24 patients, 67% were recruited from the emergency department and the remainder were recruited from the intensive care unit (21%), paediatric department (8%) and maternity department (4%). Informed verbal consent was obtained from patients and/or their parents before blood collection. Assessments of P. falciparum susceptibility to anti-malarial drugs were performed with the same venous blood sample used for diagnostic purposes. The study was reviewed and approved by the ethical committee of the Hôpital Principal de Dakar. Patients were successfully treated by QN.

Thin blood smears were stained using a RAL® kit (Réactifs RAL, Paris, France) based on eosin and methylene blue and were examined to determine P. falciparum density and to confirm monoinfection. Parasitized erythrocytes were washed three times in RPMI 1640 medium (Invitrogen, Paisley, UK) buffered with 25 mM HEPES and 25 mM NaHCO3. If parasitaemia exceeded 0.5%, infected erythrocytes were diluted to 0.5% with uninfected erythrocytes (human blood type A+) and resuspended in RPMI 1640 medium supplemented with 10% human serum (Abcys S.A. Paris, France), for a final haematocrit of 1.5%.

Drugs

CQ, QN and DHA were purchased from Sigma (Saint Louis, MO, USA). MDAQ was obtained from the World Health Organization (Geneva, Switzerland), MQ was purchased from Roche (Paris, France), and LMF was purchased from Novartis Pharma (Basel, Switzerland). AS, PPQ and PND were obtained from Shin Poong Pharm Co (Seoul, Korea) and PVB from Provepharm SAS (Marseille, France). QN, MDAQ, MQ, DHA, AS, and PPQ were first dissolved in methanol and then diluted in water to final concentrations ranging from 6 nM to 3,149 nM for QN, 1.9 to 1,988 nM for MDAQ, 1.5 to 392 nM for MQ, 0.1 to 107 nM for DHA, 0.1 to 98 nM for AS and 1.9 to 998 nM for PPQ. CQ, PND and PVB were resuspended and diluted in water to final concentrations ranging from 6 nM to 3,231 nM, 0.4 to 199 nM and 0.5 to 500 nM, respectively. LMF was resuspended and diluted in ethanol to obtain final concentrations ranging from 0.6 nM to 310 nM.

The batches of plates were tested and validated on the CQ-susceptible 3D7 strain (West-Africa) and the CQ-resistant W2 strain (Indochina) (MR4, Virginia, USA) in three to six independent experiments using the same conditions described in the paragraph below. The two strains were synchronized twice with sorbitol before use [5], and clonality was verified every 15 days using PCR genotyping of the polymorphic genetic markers msp1 and msp2 and microsatellite loci [6,7] and annually by an independent laboratory from the Worldwide Anti-malarial Resistance Network (WWARN).

Ex vivo assay

For the in vitro isotopic microtests, 100 μl of synchronous parasitized red blood cells (final parasitaemia, 0.5%; final haematocrit, 1.5%) was aliquoted into 96-well plates pre-dosed with anti-malarial drugs. The plates were incubated in a sealed bag for 72 hrs at 37°C with the atmospheric generators for capnophilic bacteria, Genbag CO2® at 5% CO2 and 15% O2 (BioMérieux, Marcy l’Etoile, France) [8]. After thawing the plates, haemolysed cultures were homogenized by vortexing the plates. Both the success of the drug susceptibility assay and the appropriate volume of haemolysed culture to use for each assay were determined for each clinical isolate during a preliminary HRP2 ELISA. Both the pre-test and subsequent ELISAs were performed using a commercial kit (Malaria Ag Celisa, ref KM2159, Cellabs PTY LTD, Brookvale, Australia) according to the manufacturer’s recommendations. The optical density (OD) of each sample was measured with a spectrophotometer (Multiskan EX, Thermo Scientific, Vantaa, Finland).

The concentration at which the drugs were able to inhibit 50% of parasite growth (IC50) was calculated with the inhibitory sigmoid Emax model, with estimation of the IC50 through non-linear regression using a standard function of the R software (ICEstimator version 1.2) [9]. IC50 values were validated only if the OD ratio (OD at concentration 0/OD at concentration max) was greater than 1.6 and the confidence interval ratio (upper 95% confidence interval of the IC50 estimation/lower 95% confidence interval of the IC50 estimation) was less than 2.0 [9].

Data and statistical analysis

IC50 values were analysed after logarithmic transformation and expressed as the geometric mean of the IC50 and a confidence interval of 95% (CI95%).

Using the Genbag conditions, the cut-off values for in vitro resistance, or reduced susceptibility, were 77 nM, 61 nM, 115 nM, 12 nM, 12 nM, 611 nM, and 30 nM for CQ, MDAQ, LMF, DHA, AS, QN, and MQ, respectively [10].

Results

Of the 24 patients recruited at the Hôpital Principal de Dakar, 19 were tested ex vivo, and 18 isolates were successfully cultured. The average parameter estimates for the ten anti-malarial drugs used against the P. falciparum isolates are given in Table 1. The prevalence of P. falciparum isolates with decreased susceptibility to MQ in vitro reached 55.6%. Fifty per cent of the isolates were resistant in vitro to CQ.

Table 1 Ex vivo susceptibility of 18 Plasmodium falciparum isolates from Dakar to chloroquine (CQ), monodesethylamodiaquine (MDAQ), lumefantrine (LMF), dihydroartemisinin (DHA), quinine (QN), mefloquine (MQ), artesunate (AS), pyronardine (PND), piperaquine (PPQ) and Proveblue (PVB)

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 IC50 = 30.7 nM) to 2011 (median IC50 = 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 IC50 values can be found for artemisinin, with an IC50 > 30 nM in Dakar [33] and AS with an IC50 > 45 nM [13]. The median IC50 values increased from 2008 to 2011 (3.2 to 10.1 nM) with a highest IC50 value of 73.1 nM in Thies [4]. In the present study, IC50 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 IC50 values (geometric mean IC50 = 32.2 nM) observed in Dakar in 2013 were slightly lower than those found in other ex vivo studies in Africa (geometric mean IC50 = 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 IC50 values (geometric mean IC50 = 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 IC50s 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 IC50 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.

References

  1. 1.

    Ndiaye JLA, Faye B, Gueye A, Tine R, Ndiaye D, Tchania C, et al. Repeated treatment of recurrent uncomplicated Plasmodium falciparum malaria in Senegal with fixed-dose artesunate plus amodiaquine versus fixed-dose artemether plus lumefantrine: a randomized, open-label trial. Malar J. 2011;10:237.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  2. 2.

    Thiam S, Thior M, Faye B, Diouf ML, Diouf MB, Diallo I, et al. Major reduction in anti-malarial drug consumption in Senegal after nation-wide introduction of malaria rapid diagnostic tests. PLoS One. 2011;6:18419.

    Article  Google Scholar 

  3. 3.

    Fall B, Pascual A, Sarr FD, Wurtz N, Richard V, Baret E, et al. Plasmodium falciparum susceptibility to anti-malarial drugs in Dakar, Senegal: an ex vivo and drug resistance molecular markers study. Malar J. 2013;12:107.

    Article  PubMed Central  PubMed  Google Scholar 

  4. 4.

    Van Tyne D, Dieye B, Valim C, Daniels RF, Diogoye Sène P, Lukens AK, et al. Changes in drug sensitivity and anti-malarial drug resistance mutations over time among Plasmodium falciparum parasites in Senegal. Malar J. 2013;12:441.

    Article  PubMed Central  PubMed  Google Scholar 

  5. 5.

    Lambros C, Vanderberg JP. Synchronization of Plasmodium falciparum erythrocytic stages in culture. J Parasitol. 1979;65:418–20.

    Article  CAS  PubMed  Google Scholar 

  6. 6.

    Bogreau H, Renaud F, Bouchiba H, Durand P, Assi SB, Henry MC, et al. Genetic diversity and structure of African Plasmodium falciparum populations in urban and rural areas. Am J Trop Med Hyg. 2006;74:953–9.

    CAS  PubMed  Google Scholar 

  7. 7.

    Henry M, Diallo I, Bordes J, Ka S, Pradines B, Diatta B, et al. Urban malaria in Dakar, Senegal: chemosusceptibility and genetic diversity of Plasmodium falciparum isolates. Am J Trop Med Hyg. 2006;75:146–51.

    CAS  PubMed  Google Scholar 

  8. 8.

    Pascual A, Basco LK, Baret E, Amalvict R, Travers D, Rogier C, et al. Use of the atmospheric generators for capnophilic bacteria Genbag CO2® for the evaluation of in vitro Plasmodium falciparum susceptibility to standard anti-malarial drugs. Malar J. 2011;10:8.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  9. 9.

    Le Nagard H, Vincent C, Mentré F, Le Bras J. Online analysis of in vitro resistance to antimalarial drugs through nonlinear regression. Comput Methods Programs Biomed. 2011;104:10–8.

    Article  PubMed  Google Scholar 

  10. 10.

    Fall B, Diawara S, Sow K, Baret E, Diatta B, Fall KB, et al. Ex vivo susceptibility of Plasmodium isolates from Dakar, Senegal, to seven standard anti-malarial drugs. Malar J. 2011;10:310.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  11. 11.

    Drame PM, Machault V, Diallo A, Cornélie S, Poinsignon A, Lalou R, et al. IgG responses to the gSG6-P1 salivary peptide for evaluating human exposure to Anopheles bites in urban areas of Dakar. Senegal Malar J. 2012;11:72.

    Article  CAS  Google Scholar 

  12. 12.

    Trape JF, Tall A, Sokhna C, Badara Ly A, Diagne N, Ndiath O, et al. The rise and fall of malaria in a west African rural community, Dielmo, Senegal, from 1990 to 2012: a 22 year longitudinal study. Lancet Infect Dis. 2014;14:476–88.

    Article  PubMed  Google Scholar 

  13. 13.

    Jambou R, Legrand E, Niang M, Khim N, Lim P, Volney B, et al. Resistance of Plasmodium falciparum field isolates to in vitro artemether and point mutations of the SERCA-type PfATPase6. Lancet. 2005;366:1960–3.

    Article  CAS  PubMed  Google Scholar 

  14. 14.

    Gari-Toussaint M, Pradines B, Mondain V, Keundjian A, Dellamonica P, Le Fichoux Y. Sénégal et paludisme. Echec prophylactique vrai de la méfloquine. Presse Med. 2002;31:1136.

    CAS  PubMed  Google Scholar 

  15. 15.

    Hatin I, Trape JF, Legros F, Bauchet J, Le Bras J. Susceptibility of Plasmodium falciparum strains to mefloquine in an urban area in Senegal. Bull World Health Organ. 1992;70:363–7.

    PubMed Central  CAS  PubMed  Google Scholar 

  16. 16.

    Sokhna C, Molez JF, Ndiaye P, Sane B, Trape JF. Tests in vivo de chimiosensibilite de Plasmodium falciparum à la chloroquine au Sénégal: évolution de la résistance et estimation de l’efficacité thérapeutique. Bull Soc Pathol Exot. 1997;90:83–9.

    CAS  PubMed  Google Scholar 

  17. 17.

    Thomas SM, Ndir O, Dieng T, Mboup S, Wypij D, Maguire JH, et al. In vitro chloroquine susceptibility and PCR analysis of pfcrt and pfmdr1 polymorphisms in Plasmodium falciparum isolates from Senegal. Am J Trop Med Hyg. 2002;66:474–80.

    CAS  PubMed  Google Scholar 

  18. 18.

    Sarr O, Myrick A, Daily J, Diop BM, Dieng T, Ndir O, et al. In vivo and in vitro analysis of chloroquine resistance in Plasmodium falciparum isolates from Senegal. Parasitol Res. 2005;97:136–40.

    Article  PubMed Central  PubMed  Google Scholar 

  19. 19.

    Ly O, Gueye PE, Deme AB, Dieng T, Badiane AS, Ahouidi AD, et al. Evolution of the pfcrt T76 and pfmdr1 Y86 markers and chloroquine susceptibility 8 years after cessation of chloroquine use in Pikine, Senegal. Parasitol Res. 2012;111:1541–6.

    Article  PubMed  Google Scholar 

  20. 20.

    Sarr O, Ahouidi AD, Ly O, Daily JP, Ndiaye D, Ndir O, et al. Mutations in PfCRT K76T do no correlate with sulfadoxine-pyrimethamine-amodiaquine failure in Pikine. Parasitol Res. 2008;103:765–9.

    Article  PubMed Central  PubMed  Google Scholar 

  21. 21.

    Gharbi M, Flegg JA, Hubert V, Kendjo E, Metcalf JE, Bertaux L, et al. Longitudinal study assessing the return of chloroquine susceptibility of Plasmodium falciparum in isolates from travellers returning from West and Central Africa, 2000–2011. Malar J. 2013;12:35.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  22. 22.

    Ministère de la Santé et de la Prévention Médicale. Enquête nationale sur le paludisme au Sénégal 2006. 2007. http://www.measuredhs.com/pubs/pdf/MIS1/MIS1.pdf.

  23. 23.

    Ministère de la Santé et de la Prévention Médicale. Enquête nationale sur le paludisme au Sénégal 2008–2009. 2009. http://www.measuredhs.com/pubs/pdf/MIS5/MIS5.pdf [revised30Sep2009].

  24. 24.

    Noranate N, Durand R, Tall A, Marrama L, Spiegel A, Sokhna C, et al. Rapid dissemination of Plasmodium falciparum drug resistance despite strictly controlled antimalarial use. Plos One. 2007;1:139.

    Article  Google Scholar 

  25. 25.

    Brasseur P, Guiguemde R, Diallo S, Guiyedi V, Kombila M, Ringwald P, et al. Amodiaquine remains effective for treating ucomplicated malaria in West and Central Africa. Trans R Soc Trop Med Hyg. 1999;93:645–50.

    Article  CAS  PubMed  Google Scholar 

  26. 26.

    Ndiaye JL, Randrianarivelojosia M, Sagara I, Brasseur P, Ndiaye I, Faye B, et al. Randomized, multicentre assessment of the efficacy and safety of ASAQ – a fixed dose artesunate-amodiaquine combination therapy in the treatment of uncomplicated Plasmodium falciparum malaria. Malar J. 2009;8:125.

    Article  PubMed Central  PubMed  Google Scholar 

  27. 27.

    Dondorp AM, Nosten F, Yi P, Das D, Phyo AP, Tarning J, et al. Artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med. 2009;361:455–67.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  28. 28.

    Noedl H, Se Y, Schaecher K, Smith BL, Socheat D, Fukuda MM. Evidence of artemisinin-resistant malaria in western Cambodia. N Engl J Med. 2008;359:2619–20.

    Article  CAS  PubMed  Google Scholar 

  29. 29.

    Phyo AP, Nkhoma S, Stepniewska K, Ashley EA, Nair S, McGready R, et al. Emergence of artemisinin-resistant malaria on the western border of Thailand: a longitudinal study. Lancet. 2012;379:1960–6.

    Article  PubMed Central  PubMed  Google Scholar 

  30. 30.

    Ashley EA, Dhorda M, Fairhurst RM, Amaratunga C, Lim P, Suon S, et al. Spread of artemisinin resistance in Plasmodium falciparum malaria. N Eng J Med. 2014;371:411–23.

    Article  CAS  Google Scholar 

  31. 31.

    Pradines B, Tall A, Rogier C, Spiegel A, Mosnier J, Marrama L, et al. In vitro activities of ferrochloroquine against 55 Senegalese isolates of Plasmodium falciparum in comparison with those of standard antimalarial drugs. Trop Med Int Health. 2002;7:265–70.

    Article  CAS  PubMed  Google Scholar 

  32. 32.

    Pradines B, Mabika Mamfoumbi M, Tall A, Sokhna C, Koeck JL, Fusai T, et al. In vitro activity of tafenoquine against the asexual blood stages of Plasmodium falciparum isolates from Gabon, Senegal, and Djibouti. Antimicrob Agents Chemother. 2006;50:3225–6.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  33. 33.

    Ndiaye D, Patel V, Demas A, LeRoux M, Ndir O, Mboup S, et al. An non-radioactive DAPI-based high-throughput in vitro assay to assess Plasmodium falciparum responsiveness to antimalarials – Increased sensitivity of P. falciparum to chloroquine in Senegal. Am J Trop Med Hyg. 2010;82:228–30.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  34. 34.

    Witkowski B, Amaratunga C, Khim N, Sreng S, Chim P, Kim S, et al. Novel phenotypic assays for the detection of artemisinin-resistant Plasmodium falciparum malaria in Cambodia: in-vitro and ex-vivo drug-response studies. Lancet Infect Dis. 2013;13:1043–9.

    Article  CAS  PubMed  Google Scholar 

  35. 35.

    Witkowski B, Khim N, Chim P, Kim S, Ke S, Kloeung N, et al. Reduced artemisinin susceptibility of Plasmodium falciparum ring stages in Western Cambodia. Antimicrob Agents Chemother. 2013;57:914–23.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  36. 36.

    Ariey F, Witkowsky B, Amaratunga C, Beghain J, Langlois AC, Khim N, et al. A molecular marker of artemisinin-resistant Plasmodium falciparum. Nature. 2014;505:50–5.

    Article  PubMed  Google Scholar 

  37. 37.

    Amaratunga C, Witkowski B, Khim N, Menard D, Fairhurst RM. Artemisinin resistance in Plasmodium falciparum. Lancet Infect Dis. 2014;14:449–50.

    Article  PubMed  Google Scholar 

  38. 38.

    Yavo W, Faye B, Kuete T, Djohan V, Oga SA, Kassi RR, et al. Multicentric assessment of the efficacy and tolerability of dihydroartemisinin-piperaquine compared to artemether-lumefantrine in the treatment of uncomplicated Plasmodium falciparum malaria in sub-Saharan Africa. Malar J. 2011;10:198.

    Article  PubMed Central  PubMed  Google Scholar 

  39. 39.

    Yeka A, Tibenderana J, Achan J, D’Alessandro U, Talisuna AO. Efficacy of quinine, artemether-lumefantrine and dihydroartemisinin-piperaquine as rescue treatment for uncomplicated malaria in Ugandan children. PLoS One. 2013;8:53772.

    Article  Google Scholar 

  40. 40.

    Agarwal A, McMorrow M, Onyango P, Otieno K, Odero C, Williamson J, et al. A randomized trial of artemether-lumefantrine and dihydroartemisinin-piperaquine in the treatment of uncomplicated malaria among children in western Kenya. Malar J. 2013;12:254.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  41. 41.

    Pascual A, Madamet M, Bertaux L, Amalvict R, Benoit N, Travers D, et al. In vitro piperaquine susceptibility is not associated with the Plasmodium falciparum chloroquine resistance transporter gene. Malar J. 2013;12:431.

    Article  PubMed Central  PubMed  Google Scholar 

  42. 42.

    Pascual A, Parola P, Benoit-Vical F, Simon F, Malvy D, Picot S, et al. Ex vivo activity of the ACT new components pyronaridine and piperaquine in comparison with conventional ACT drugs against isolates of Plasmodium falciparum. Malar J. 2012;11:45.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  43. 43.

    Brandicourt O, Druilhe P, Diouf F, Brasseur P, Turk P, Danis M. Decreased sensitivity to chloroquine and quinine of some Plasmodium falciparum strains from Senegal in september 1984. Am J Trop Med Hyg. 1986;35:717–21.

    CAS  PubMed  Google Scholar 

  44. 44.

    Pradines B, Tall A, Parzy D, Spiegel A, Fusai T, Hienne R, et al. In vitro activity of pyronaridine and amodiaquine against African isolates (Senegal) of Plasmodium falciparum in comparison with standard antimalarial agents. J Antimicrob Chemother. 1998;42:333–9.

    Article  CAS  PubMed  Google Scholar 

  45. 45.

    Pradines B, Tall A, Ramiandrasoa F, Spiegel A, Sokhna C, Fusai T, et al. In vitro activity of iron-binding compounds against Senegalese isolates of Plasmodium falciparum. J Antimicrob Chemother. 2006;57:1093–9.

    Article  CAS  PubMed  Google Scholar 

  46. 46.

    Jelinek T, Schelbert P, Loscher T, Eichenlaub D. Quinine resistant falciparum malaria acquired in east Africa. Trop Med Parasitol. 1995;46:38–40.

    CAS  PubMed  Google Scholar 

  47. 47.

    Palmieri F, Petrosillo N, Paglia MG, Conte A, Goletti D, Pucillo LP, et al. Genetic confirmation of quinine-resistant Plasmodium falciparum malaria followed by postmalaria neurological syndrome in a traveler from Mozambique. J Clin Microbiol. 2004;42:5424–6.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  48. 48.

    Pradines B, Pistone T, Ezzedine K, Briolant S, Bertaux L, Receveur MC, et al. Quinine-resistant malaria in traveler returning from Senegal, 2007. Emerg Infect Dis. 2010;16:546–8.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  49. 49.

    Pascual A, Henry M, Briolant S, Charras S, Baret E, Amalvict R, et al. In vitro activity of Proveblue (methylene blue) on Plasmodium falciparum strains resistant to standard antimalarial drugs. Antimicrob Agents Chemother. 2011;55:2472–4.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  50. 50.

    Dormoi J, Pascual A, Briolant S, Amalvict R, Charras S, Baret E, et al. Proveblue (methylene blue) as antimalarial agent: in vitro synergy with dihydroartemisinin and atorvastatin. Antimicrob Agents Chemother. 2012;56:3467–9.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  51. 51.

    Dormoi J, Briolant S, Desgrouas C, Pradines B. Efficacy of Proveblue (methylene blue) in an experimental cerebral murine model. Antimicrob Agents Chemother. 2013;57:3412–4.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  52. 52.

    Dormoi J, Briolant S, Desgrouas C, Pradines B. Impact of methylene blue and atorvastatin combination therapy on the apparition of cerebral malaria in a murine model. Malar J. 2013;12:127.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  53. 53.

    Dormoi J, Pradines B. Dose responses of Proveblue methylene blue in an experimental murine cerebral malaria model. Antimicrob Agents Chemother. 2013;57:4080–1.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  54. 54.

    Ademowo OG, Nneji CM, Adedapo AD. In vitro antimalarial activity of methylene blue against field isolates of Plasmodium falciparum from children in Southeast Nigeria. Indian J Med Res. 2007;126:45–9.

    CAS  PubMed  Google Scholar 

  55. 55.

    Okombo J, Kiara SM, Mwai L, Pole L, Ohuma E, Ochola LI, et al. Baseline of the activities of the antimalarials pyronaridine and methylene blue against Plasmodium falciparum isolates from Kenya. Antimicrob Agents Chemother. 2012;56:1105–7.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  56. 56.

    Suwanarusk R, Russel B, Ong A, Sriprawat K, Chu CS, Phyo AP, et al. Methylene blue inhibits the asexual development of vivax malaria parasites from a region of increasing chloroquine resistance. J Antimicrob Chemother. 2014. in press.

  57. 57.

    Adjalley SH, Jonhston GL, Li T, Eastman RT, Ekland EH, Eappen AG, et al. Quantitative assessment of Plasmodium falciparum sexual development revels potent transmission-blocking activity by methylene blue. Proc Natl Acad Sci U S A. 2011;108:1214–23.

    Article  Google Scholar 

  58. 58.

    Coulibaly B, Zoungrana A, Mockenhaupt FP, Schirmer RH, Klose C, Mansmann U, et al. Strong gametocytocidal effect of methylene blue-based combination therapy against falciparum malaria: a randomized control trial. PLoS One. 2009;4:5318.

    Article  Google Scholar 

Download references

Acknowledgements

The authors thank the patients and the staff of the Hôpital Principal de Dakar. The authors thank Ndeye Fatou Diop and Maurice Gomis from the Hôpital Principal de Dakar for technical support.

This study was supported by the Schéma directeur Paludisme, Etat Major des Armées Françaises (grant LR 607a) and by the Ministère des Affaires Etrangères.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Bruno Pradines.

Additional information

Competing interests

The authors declare that they have no competing interests.

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.

Rights and permissions

Open Access  This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.

The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

To view a copy of this licence, visit https://creativecommons.org/licenses/by/4.0/.

The Creative Commons Public Domain Dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Fall, B., Camara, C., Fall, M. et al. Plasmodium falciparum susceptibility to standard and potential anti-malarial drugs in Dakar, Senegal, during the 2013–2014 malaria season. Malar J 14, 60 (2015). https://doi.org/10.1186/s12936-015-0589-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12936-015-0589-3

Keywords

  • Malaria
  • Plasmodium falciparum
  • Anti-malarial
  • In vitro
  • Resistance
  • Senegal
  • Proveblue
  • Methylene blue