Association between Polymorphisms in the Pfmdr6 Gene and Ex Vivo Susceptibility to Quinine in Plasmodium falciparum Parasites from Dakar, Senegal.

Polymorphisms and the overexpression of transporter genes, especially of the ATP-binding cassette superfamily, have been involved in antimalarial drug resistance. The objective of this study was to use 77 Senegalese Plasmodium falciparum isolates to evaluate the association between the number of Asn residues in the polymorphic microsatellite region of the Plasmodium falciparum multidrug resistance 6 gene (Pfmdr6) and the ex vivo susceptibility to antimalarials. A significant association was observed between the presence of 7 or 9 Asn repeats and reduced susceptibility to quinine.

Prevalence of anti-malarial resistance genes in Dakar, Senegal from 2013 to 2014.

BACKGROUND: To determine the impact of the introduction of artemisinin-based combination therapy (ACT) on parasite susceptibility, a molecular surveillance for antimalarial drug resistance was conducted on local isolates from the Hôpital Principal de Dakar between November 2013 and January 2014 and between August 2014 and December 2014. METHODS: The prevalence of genetic polymorphisms in antimalarial resistance genes (pfcrt, pfmdr1, pfdhfr and pfdhps) was evaluated in 103 isolates. RESULTS: The chloroquine-resistant haplotypes CVIET and CVMET were identified in 31.4 and 3.9 % of the isolates, respectively. The frequency of the pfcrt K76T mutation was increased from 29.3 % in 2013-2014 to 43.2 % in 2014. The pfmdr1 N86Y and Y184F mutations were identified in 6.1 and 53.5 % of the isolates, respectively. The pfdhfr triple mutant (S108N, N51I and C59R) was detected in the majority of the isolates (82.3 %). The prevalence of quadruple mutants (pfdhfr S108N, N51I, C59R and pfdhps A437G) was 40.4 %. One isolate (1.1 %) harboured the pfdhps mutations A437G and K540E and the pfdhfr mutations S108N, N51I and C59R. CONCLUSIONS: Despite a decline in the prevalence of chloroquine resistance due to the official withdrawal of the drug and to the introduction of ACT, the spread of resistance to chloroquine has continued. Furthermore, susceptibility to amodiaquine may be decreased as a result of cross-resistance. The frequency of the pfmdr1 mutation N86Y declined while the Y184F mutation increased in prevalence, suggesting that selective pressure is acting on pfmdr1, leading to a high prevalence of mutations in these isolates and the lack of specific mutations. The 50.5 % prevalence of the pfmdr1 polymorphisms N86Y and Y184F suggests a decrease in lumefantrine susceptibility. Based on these results, intensive surveillance of ACT partner drugs must be conducted regularly in Senegal.

Absence of Association between Polymorphisms in the RING E3 Ubiquitin Protein Ligase Gene and Ex Vivo Susceptibility to Conventional Antimalarial Drugs in Plasmodium falciparum Isolates from Dakar, Senegal.

The RING E3 ubiquitin protein ligase is crucial for facilitating the transfer of ubiquitin. The only polymorphism identified in the E3 ubiquitin protein ligase gene was the D113N mutation (62.5%) but was not significantly associated with the 50% inhibitory concentration (IC50) of conventional antimalarial drugs. However, some mutated isolates (D113N) present a trend of reduced susceptibility to piperaquine (P = 0.0938). To evaluate the association of D113N polymorphism with susceptibility to antimalarials, more isolates are necessary.

Plasmodium falciparum In Vitro Resistance to Monodesethylamodiaquine, Dakar, Senegal, 2014.

We successfully cultured 36 Plasmodium falciparum isolates from blood samples of 44 malaria patients admitted to the Hôpital Principal de Dakar (Dakar, Senegal) during August-December 2014. The prevalence of isolates with in vitro reduced susceptibility was 30.6% for monodesethylamodiaquine, 52.8% for chloroquine, 44.1% for mefloquine, 16.7% for doxycycline, 11.8% for piperaquine, 8.3% for artesunate, 5.9% for pyronaridine, 2.8% for quinine and dihydroartemisinin, and 0.0% for lumefantrine. The prevalence of isolates with reduced in vitro susceptibility to the artemisinin-based combination therapy partner monodesethylamodiaquine increased from 5.6% in 2013 to 30.6% in 2014. Because of the increased prevalence of P. falciparum parasites with impaired in vitro susceptibility to monodesethylamodiaquine, the implementation of in vitro and in vivo surveillance of all artemisinin-based combination therapy partners is warranted.

Emergence of Mutations in the K13 Propeller Gene of Plasmodium falciparum Isolates from Dakar, Senegal, in 2013-2014.

The kelch 13 (K13) propeller gene is associated with artemisinin resistance. In a previous work, there were no mutations found in 138 Plasmodium falciparum isolates collected in 2012 and 2013 from patients residing in Dakar, Senegal (M. Torrentino-Madamet et al., Malar J 13:472, 2014, http://dx.doi.org/10.1186/1475-2875-13-472). However, the N554H, Q613H, and V637I mutations were identified in the propeller region of K13 in 92 (5.5%) isolates in 2013 and 2014. There were five polymorphisms identified in the Plasmodium/Apicomplexa-specific domain (K123R, N137S, N142NN/NNN, T149S, and K189T/N).

MALDI-TOF Mass Spectrometry: A Powerful Tool for Clinical Microbiology at Hôpital Principal de Dakar, Senegal (West Africa).

Our team in Europe has developed the routine clinical laboratory identification of microorganisms by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). To evaluate the utility of MALDI-TOF MS in tropical Africa in collaboration with local teams, we installed an apparatus in the Hôpital Principal de Dakar (Senegal), performed routine identification of isolates, and confirmed or completed their identification in France. In the case of discordance or a lack of identification, molecular biology was performed. Overall, 153/191 (80.1%) and 174/191 (91.1%) isolates yielded an accurate and concordant identification for the species and genus, respectively, with the 2 different MALDI-TOF MSs in Dakar and Marseille. The 10 most common bacteria, representing 94.2% of all bacteria routinely identified in the laboratory in Dakar (Escherichia coli, Klebsiella pneumoniae, Streptococcus agalactiae, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus haemolyticus, Enterobacter cloacae, Enterococcus faecalis, and Staphylococcus epidermidis) were accurately identified with the MALDI-TOF MS in Dakar. The most frequent misidentification in Dakar was at the species level for Achromobacter xylosoxidans, which was inaccurately identified as Achromobacter denitrificans, and the bacteria absent from the database, such as Exiguobacterium aurientacum or Kytococcus schroeteri, could not be identified. A few difficulties were observed with MALDI-TOF MS for Bacillus sp. or oral streptococci. 16S rRNA sequencing identified a novel bacterium, "Necropsobacter massiliensis." The robust identification of microorganisms by MALDI-TOF MS in Dakar and Marseille demonstrates that MALDI-TOF MS can be used as a first-line tool in clinical microbiology laboratories in tropical countries.

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

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.

The ongoing revolution of MALDI-TOF mass spectrometry for microbiology reaches tropical Africa.

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) represents a revolution in routine pathogen identification in clinical microbiology laboratories. A MALDI-TOF MS was introduced to tropical Africa in the clinical microbiology laboratory of the Hôpital Principal de Dakar (Senegal) and used for routine pathogen identification. Using MS, 2,429 bacteria and fungi isolated from patients were directly assayed, leading to the identification of 2,082 bacteria (85.7%) and 206 fungi (8.5%) at the species level, 109 bacteria (4.5%) at the genus level, and 16 bacteria (0.75%) at the family level. Sixteen isolates remained unidentified (0.75%). Escherichia coli was the most prevalent species (25.8%) followed by Klebsiella pneumoniae (14.8%), Streptococcus agalactiae (6.2%), Acinetobacter baumannii (6.1%), Pseudomonas aeruginosa (5.9%), and Staphylococcus aureus (5.9%). MALDI-TOF MS has also enabled the detection of rare bacteria and fungi. MALDI-TOF MS is a powerful tool for the identification of bacterial and fungal species involved in infectious diseases in tropical Africa.

Limited polymorphisms in k13 gene in Plasmodium falciparum isolates from Dakar, Senegal in 2012-2013.

BACKGROUND: The emergence of Plasmodium falciparum resistance to artemisinin and its derivatives, manifested as delayed parasite clearance following the treatment, has developed in Southeast Asia. The spread of resistance to artemisinin from Asia to Africa may be catastrophic for malaria control and elimination worldwide. Recently, mutations in the propeller domain of the Kelch 13 (k13) gene (PF3D71343700) were associated with in vitro resistance to artemisinin and with delayed clearance after artemisinin treatment in southern Asia. The aim of the study was to characterize the genetic variability of k13 and to evaluate the molecular resistance to artemisinin for the first time in Senegal. METHODS: Plasmodium falciparum isolates were collected from 138 malaria patients in Dakar and its districts during the rainy season of October 2012 to January 2013 at the Hôpital Principal de Dakar. The k13 gene was amplified using nested PCR and sequenced. RESULTS: A very limited variability within the k13 gene in Senegalese P. falciparum isolates was identified. No polymorphism was detected in the six k13-propeller blades. Only two mutations, T149S (6.3%) and K189T (42.2%), and one (N) or two (NN) asparagine insertion at the codon 142 (4.7 and 6.3%, respectively) were detected in the Plasmodium/Apicomplexa-specific domain. None of the polymorphisms associated with artemisinin resistance in Southeast Asia was detected in the 138 P. falciparum from Dakar. DISCUSSION: The present data do not suggest widespread artemisinin resistance in Dakar in 2012-2013. Notably, the C580Y, R539T or Y493H substitutions that were associated with in vitro resistance or delayed parasite clearance in Southeast Asia were not observed in Dakar, nor were any of the polymorphisms observed in parasites from Southeast Asia, nor the M476I mutation that was selected in vitro with artemisinin pressure in a African parasite line.

Role of Pfmdr1 in in vitro Plasmodium falciparum susceptibility to chloroquine, quinine, monodesethylamodiaquine, mefloquine, lumefantrine, and dihydroartemisinin.

The involvement of Pfmdr1 (Plasmodium falciparum multidrug resistance 1) polymorphisms in antimalarial drug resistance is still debated. Here, we evaluate the association between polymorphisms in Pfmdr1 (N86Y, Y184F, S1034C, N1042D, and D1246Y) and Pfcrt (K76T) and in vitro responses to chloroquine (CQ), mefloquine (MQ), lumefantrine (LMF), quinine (QN), monodesethylamodiaquine (MDAQ), and dihydroartemisinin (DHA) in 174 Plasmodium falciparum isolates from Dakar, Senegal. The Pfmdr1 86Y mutation was identified in 14.9% of the samples, and the 184F mutation was identified in 71.8% of the isolates. No 1034C, 1042N, or 1246Y mutations were detected. The Pfmdr1 86Y mutation was significantly associated with increased susceptibility to MDAQ (P = 0.0023), LMF (P = 0.0001), DHA (P = 0.0387), and MQ (P = 0.00002). The N86Y mutation was not associated with CQ (P = 0.214) or QN (P = 0.287) responses. The Pfmdr1 184F mutation was not associated with various susceptibility responses to the 6 antimalarial drugs (P = 0.168 for CQ, 0.778 for MDAQ, 0.324 for LMF, 0.961 for DHA, 0.084 for QN, and 0.298 for MQ). The Pfmdr1 86Y-Y184 haplotype was significantly associated with increased susceptibility to MDAQ (P = 0.0136), LMF (P = 0.0019), and MQ (P = 0.0001). The additional Pfmdr1 86Y mutation increased significantly the in vitro susceptibility to MDAQ (P < 0.0001), LMF (P < 0.0001), MQ (P < 0.0001), and QN (P = 0.0026) in wild-type Pfcrt K76 parasites. The additional Pfmdr1 86Y mutation significantly increased the in vitro susceptibility to CQ (P = 0.0179) in Pfcrt 76T CQ-resistant parasites.

In vitro susceptibility to quinine and microsatellite variations of the Plasmodium falciparum Na+/H+ exchanger transporter (Pfnhe-1) gene in 393 isolates from Dakar, Senegal.

BACKGROUND: Although the World Health Organization recommends replacing quinine (QN) by artesunate due to its increased efficacy and the higher tolerance to the drug in both adults and children, QN remains a first-line treatment for severe malaria, especially in Africa. Investigations of microsatellite Pfnhe-1 ms4760 polymorphisms in culture-adapted isolates from around the world have revealed that an increase in the number of DNNND amino acid motifs was associated with decreased QN susceptibility, whereas an increase in the number of DDNHNDNHNND motifs was associated with increased QN susceptibility. METHODS: In this context, to further analyse associations between Pfnhe-1 ms4760 polymorphisms and QN susceptibility, 393 isolates freshly collected between October 2009 and January 2010 and July 2010 and February 2011, respectively, at the Hôpital Principal de Dakar, Senegal were assessed ex vivo for QN susceptibility, and their genes were amplified and sequenced. RESULTS: Of the 393 Plasmodium falciparum clinical isolates collected, 145 were successfully cultured. The 145 QN IC50s ranged from 2.1 to 1291 nM, and 17 isolates (11.7%) exceed the QN reduced susceptibility threshold of 611 nM. Among the 393 P. falciparum clinical isolates, 47 different alleles were observed. The three most prevalent profiles were ms4760-1 (no = 72; 18.3%), ms4760-3 (no = 65; 16.5%) and ms4760-7 (no = 40; 10.2%). There were no significant associations observed between QN IC50 values and i) the number of repeats of DNNND in block II (p = 0.0955, Kruskal-Wallis test); ii) the number of repeats of DDNHNDNHNND in block V (p = 0.1455, Kruskal-Wallis test); or iii) ms4760 profiles (p = 0.1809, Kruskal-Wallis test). CONCLUSIONS: Pfnhe-1 ms4760 was highly diverse in parasite isolates from Dakar (47 different profiles). Three profiles (ms4760-1, ms4760-3 and ms4760-7) were predominant. The number of repeats for block II (DNNND) or block V (DDNHNDNHNND) was not significantly associated with QN susceptibility. New studies, and especially in vivo studies, are necessary to confirm the role of Pfnhe-1 ms4760 as a marker of QN resistance.

Plasmodium falciparum susceptibility to anti-malarial drugs in Dakar, Senegal, in 2010: an ex vivo and drug resistance molecular markers study.

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 resistance of P. falciparum to anti-malarial drugs. To determine whether parasite susceptibility has been affected by the new anti-malarial policies, an ex vivo susceptibility and drug resistance molecular marker study was conducted on local isolates obtained from the Centre de santé Elizabeth Diouf (Médina, Dakar, Senegal). METHODS: The prevalence of genetic polymorphisms in genes associated with anti-malarial drug resistance, i.e., pfcrt, pfdhfr, pfdhps and pfmdr1, were evaluated for a panel of 165 isolates collected from patients recruited from 17 August 2010 to 6 January 2011. The malaria isolates were assessed for susceptibility to chloroquine (CQ); quinine (QN); monodesethylamodiaquine (MDAQ), the active metabolite of amodiaquine; mefloquine (MQ); lumefantrine (LMF); dihydroartemisinin (DHA), the active metabolite of artemisinin derivatives; and doxycycline (DOX) using the Plasmodium lactate dehydrogenase (pLDH) ELISA. RESULTS: The prevalence of the in vitro resistant isolates, or isolates with reduced susceptibility, was 62.1% for MQ, 24.2% for CQ, 10.3% for DOX, 11.8% MDAQ, 9.7% for QN, 2.9% for LMF and 0% for DHA. The Pfcrt 76T mutation was identified in 43.6% of the samples. The pfmdr1 86Y, 184F and 1246Y mutations were found in 16.2%, 50.0% and 1.6% of the samples, respectively. The pfdhfr 108N, 51I and 59R mutations were identified in 81.9%, 77.4% and 79.4% of the samples, respectively. The double mutant (108N and 51I) was detected in 75.5% of the isolates, and the triple mutant (108N, 51I and 59R) was detected in 73.6% of the isolates. The pfdhps 437G, 436A and 613S mutations were found in 54.4%, 38.6% and 1.2% of the samples, respectively. There was only one double mutant, 437G and 540E, and one quintuple mutant, pfdhfr 108N, 51I and 59R and pfdhps 437G and 540E. The prevalence of the quadruple mutant (pfdhfr 108N, 51I and 59R and pfdhps 437G) was 36.7%. CONCLUSIONS: The results of this study indicate that an intensive surveillance of the in vitro P. falciparum susceptibility to anti-malarial drugs must be conducted in Senegal.

Pfhrp2 and pfhrp3 polymorphisms in Plasmodium falciparum isolates from Dakar, Senegal: impact on rapid malaria diagnostic tests.

BACKGROUND: An accurate diagnosis is essential for the rapid and appropriate treatment of malaria. The accuracy of the histidine-rich protein 2 (PfHRP2)-based rapid diagnostic test (RDT) Palutop+4® was assessed here. One possible factor contributing to the failure to detect malaria by this test is the diversity of the parasite PfHRP2 antigens. METHODS: PfHRP2 detection with the Palutop+4® RDT was carried out. The pfhrp2 and pfhrp3 genes were amplified and sequenced from 136 isolates of Plasmodium falciparum that were collected in Dakar, Senegal from 2009 to 2011. The DNA sequences were determined and statistical analyses of the variation observed between these two genes were conducted. The potential impact of PfHRP2 and PfHRP3 sequence variation on malaria diagnosis was examined. RESULTS: Seven P. falciparum isolates (5.9% of the total isolates, regardless of the parasitaemia; 10.7% of the isolates with parasitaemia ≤0.005% or ≤250 parasites/µl) were undetected by the PfHRP2 Palutop+4® RDT. Low parasite density is not sufficient to explain the PfHRP2 detection failure. Three of these seven samples showed pfhrp2 deletion (2.4%). The pfhrp3 gene was deleted in 12.8%. Of the 122 PfHRP2 sequences, 120 unique sequences were identified. Of the 109 PfHRP3 sequences, 64 unique sequences were identified. Using the Baker's regression model, at least 7.4% of the P. falciparum isolates in Dakar were likely to be undetected by PfHRP2 at a parasite density of ≤250 parasites/µl (slightly lower than the evaluated prevalence of 10.7%). This predictive prevalence increased significantly between 2009 and 2011 (P = 0.0046). CONCLUSION: In the present work, 10.7% of the isolates with a parasitaemia ≤0.005% (≤250 parasites/µl) were undetected by the PfHRP2 Palutop+4® RDT (7.4% by the predictive Baker'model). In addition, all of the parasites with pfhrp2 deletion (2.4% of the total samples) and 2.1% of the parasites with parasitaemia >0.005% and presence of pfhrp2 were not detected by PfHRP2 RDT. PfHRP2 is highly polymorphic in Senegal. Efforts should be made to more accurately determine the prevalence of non-sensitive parasites to pfHRP2.

Prevalence of molecular markers of Plasmodium falciparum drug resistance in Dakar, Senegal.

BACKGROUND: As a result of the widespread resistance to chloroquine and sulphadoxine-pyrimethamine, artemisinin-based combination therapy (ACT) (including artemether-lumefantrine and artesunate-amodiaquine) has been recommended as a first-line anti-malarial regimen in Senegal since 2006. Intermittent preventive treatments with anti-malarial drugs based on sulphadoxine-pyrimethamine are also given to children or pregnant women once per month during the transmission season. Since 2006, there have been very few reports on the susceptibility of Plasmodium falciparum to anti-malarial drugs. To estimate the prevalence of resistance to several anti-malarial drugs since the introduction of the widespread use of ACT, the presence of molecular markers associated with resistance to chloroquine and sulphadoxine-pyrimethamine was assessed in local isolates at the military hospital of Dakar. METHODS: The prevalence of genetic polymorphisms in genes associated with anti-malarial drug resistance, i.e., Pfcrt, Pfdhfr, Pfdhps and Pfmdr1, and the copy number of Pfmdr1 were evaluated for a panel of 174 isolates collected from patients recruited at the military hospital of Dakar from 14 October 2009 to 19 January 2010. RESULTS: The Pfcrt 76T mutation was identified in 37.2% of the samples. The Pfmdr1 86Y and 184F mutations were found in 16.6% and 67.6% of the tested samples, respectively. Twenty-eight of the 29 isolates with the 86Y mutation were also mutated at codon 184. Only one isolate (0.6%) had two copies of Pfmdr1. The Pfdhfr 108N/T, 51I and 59R mutations were identified in 82.4%, 83.5% and 74.1% of the samples, respectively. The double mutant (108N and 51I) was detected in 83.5% of the isolates, and the triple mutant (108N, 51I and 59R) was detected in 75.3%. The Pfdhps 437G, 436F/A and 613S mutations were found in 40.2%, 35.1% and 1.8% of the samples, respectively. There was no double mutant (437G and 540E) or no quintuple mutant (Pfdhfr 108N, 51I and 59R and Pfdhps 437G and 540E). The prevalence of the quadruple mutant (Pfdhfr 108N, 51I and 59R and Pfdhps 437G) was 36.5%. CONCLUSIONS: Since 2004, the prevalence of chloroquine resistance had decreased. The prevalence of isolates with high-level pyrimethamine resistance is 83.5%. The prevalence of isolates resistant to sulphadoxine is 40.2%. However, no quintuple mutant (Pfdhfr 108N, 51I and 59R and Pfdhps 437G and 540E), which is associated with a high level of sulphadoxine-pyrimethamine resistance, has been identified to date. The resistance to amodiaquine remains moderate.

Ex vivo susceptibility of Plasmodium falciparum isolates from Dakar, Senegal, to seven standard anti-malarial drugs.

BACKGROUND: As a result of widespread chloroquine and sulphadoxine-pyrimethamine resistance, artemisinin-based combination therapy (ACT) (which includes artemether-lumefantrine and artesunate-amodiaquine) has been recommended as a first-line anti-malarial regimen in Senegal since 2006. Since then, there have been very few reports on the ex vivo susceptibility of Plasmodium falciparum to anti-malarial drugs. To examine whether parasite susceptibility has been affected by the widespread use of ACT, the ex vivo susceptibility of local isolates was assessed at the military hospital of Dakar. METHODS: The ex vivo susceptibility of 93 P. falciparum isolates from Dakar was successfully determined using the Plasmodium lactate dehydrogenase (pLDH) ELISA for the following drugs: chloroquine (CQ), quinine (QN), mefloquine (MQ), monodesethylamodiaquine (MDAQ), lumefantrine (LMF), dihydroartemisinin (DHA) and doxycycline (DOX). RESULTS: After transformation of the isolate IC50 in ratio of IC50 according to the susceptibility of the 3D7 reference strain (isolate IC50/3D7 IC50), the prevalence of the in vitro resistant isolates with reduced susceptibility was 50% for MQ, 22% for CQ, 12% for DOX, 6% for both QN and MDAQ and 1% for the drugs LMF and DHA. The highest significant positive correlations were shown between responses to CQ and MDAQ (r = 0.569; P < 0.0001), LMF and QN (r = 0.511; P < 0.0001), LMF and DHA (r = 0.428; P = 0.0001), LMF and MQ (r = 0.413; P = 0.0002), QN and DHA (r = 0.402; P = 0.0003) and QN and MQ (r = 0.421; P = 0.0001). CONCLUSIONS: The introduction of ACT in 2002 has not induced a decrease in P. falciparum susceptibility to the drugs DHA, MDAQ and LMF, which are common ACT components. However, the prevalence of P. falciparum isolates with reduced susceptibility has increased for both MQ and DOX. Taken together, these data suggest that intensive surveillance of the P. falciparum in vitro susceptibility to anti-malarial drugs in Senegal is required.