We have analyzed the profiles of 23 of Plasmodium falciparum strains for their in vitro chemosusc... more We have analyzed the profiles of 23 of Plasmodium falciparum strains for their in vitro chemosusceptibilities to piperaquine (PPQ), dihydroartemisinin (DHA), chloroquine, monodesethylamodiaquine, quinine, mefloquine, lumefantrine, atovaquone, pyrimethamine, and doxycycline (DOX) in association with polymorphisms in genes involved in quinoline resistance (Plasmodium falciparum crt [pfcrt], pfmdr1, pfmrp, and pfnhe). The 50% inhibitory concentrations (IC 50 s) for PPQ ranged from 29 to 98 nM (geometric mean ؍ 57.8 nM, 95% confidence interval [CI] ؍ 51 to 65) and from 0.4 to 5.8 nM for DHA (geometric mean ؍ 1.8 nM, 95% CI ؍ 1.4 to 2.3). We found a significant positive correlation between the responses to PPQ and DHA (r 2 ؍ 0.17; P ؍ 0.0495) and between the responses to PPQ and DOX (r 2 ؍ 0.41; P ؍ 0.001). We did not find a significant association between the PPQ IC 50 (0.0525 < P < 0.9247) or the DHA IC 50 (0.0138 < P < 0.9018) and polymorphisms in the pfcrt, pfmdr1, pfmrp, and pfnhe-1 genes. There was an absence of cross-resistance with quinolines, and the IC 50 s for PPQ and DHA were found to be unrelated to mutations in the pfcrt, pfmdr1, pfmrp, and pfnhe-1 transport protein genes, which are involved in quinoline antimalarial drug resistance. These results confirm the interest in and the efficacy of the combination of PPQ and DHA for areas in which parasites are resistant to chloroquine or other quinolines.
There is an urgent need for the discovery of new anti-malarial drugs and combination therapy. A c... more There is an urgent need for the discovery of new anti-malarial drugs and combination therapy. A combinatorial approach protects each drug from the development of resistance and reduces generally the overall transmission rate of malaria. Statins, the inhibitors of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase and a family of lipid-lowering drugs, have in vitro anti-malarial properties, and more specially atorvastatin. However, atorvastatin has a short elimination half-life (14 hours) and an efficient combination of anti-malarial drugs must associate a drug with a short elimination half-life and a drug with a long elimination half-life. The objective of the present work was to identify new potential partners among standard new anti-malarial drugs with long elimination half-life, such as lumefantrine, piperaquine, pyronaridine and atovaquone, to improve the in vitro activity of atorvastatin against different Plasmodium falciparum strains to treat uncomplicated malaria. In vitro inter...
The aim of this study was to evaluate the cultivation system in which the proper atmospheric cond... more The aim of this study was to evaluate the cultivation system in which the proper atmospheric conditions for growing Plasmodium falciparum parasites were maintained in a sealed bag. The Genbag® system associated with the atmospheric generators for capnophilic bacteria Genbag CO2® was used for in vitro susceptibility test of nine standard anti-malarial drugs and compared to standard incubator conditions. The susceptibility of 36 pre-identified parasite strains from a wide panel of countries was assessed for nine standard anti-malarial drugs (chloroquine, quinine, mefloquine, monodesethylamodiaquine, lumefantrine, dihydroartemisinin, atovaquone and pyrimethamine) by the standard 42-hour 3H-hypoxanthine uptake inhibition method using the Genbag CO2® system and compared to controlled incubator conditions (5% CO2 and 10% O2). The counts per minute values in the control wells in incubator atmospheric conditions (5% CO2 and 10% O2) were significantly higher than those of Genbag® conditions ...
ABSTRACTThe involvement ofPfmdr1(Plasmodium falciparummultidrug resistance 1) polymorphisms in an... more ABSTRACTThe involvement ofPfmdr1(Plasmodium falciparummultidrug resistance 1) polymorphisms in antimalarial drug resistance is still debated. Here, we evaluate the association between polymorphisms inPfmdr1(N86Y, Y184F, S1034C, N1042D, and D1246Y) andPfcrt(K76T) andin vitroresponses to chloroquine (CQ), mefloquine (MQ), lumefantrine (LMF), quinine (QN), monodesethylamodiaquine (MDAQ), and dihydroartemisinin (DHA) in 174Plasmodium falciparumisolates from Dakar, Senegal. ThePfmdr186Y 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. ThePfmdr186Y 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. ThePfmdr1184F mutation was not associated with various susceptibility responses to the 6 antimalarial drug...
Several studies have shown the efficacy of the intermittent preventive treatment (IPT) using sulf... more Several studies have shown the efficacy of the intermittent preventive treatment (IPT) using sulfadoxine-pyrimethamine (SP) coupled with the expanded program of immunization (EPI) in infants. However, its adoption as a strategy is conditioned by the long-term efficacy of SP. The impact of IPT-SP coupled with the EPI on the prevalence of markers of resistance to SP was evaluated during this study conducted in Southern Senegal. Three cross-sectional surveys in two health districts (IPT+) were conducted prior to the implementation, 1 year, and 2 years after. A third district located between the two districts served as a test zone (IPT-). PCR tests were carried out from filter papers collected in children under five for the two first measures and from positive rapid diagnostic tests in the same population for the third measure. Mutations in codons 51, 59, and 108 of the DHFR gene and in codons 437 and 540 of the DHPS were analyzed. The results showed that the prevalence of DHFR triple mutation was more frequent after 2 years in IPT+ areas. Regarding quadruple mutation, DHFR (51, 59, and 108) and DHPS (437), no difference was noted between the two areas. The quintuple mutation was not observed after 2 years of implementation in both areas. However, an individual analysis showed significant differences in the individual mutation points 51, 59, 108, and 437. This study reveals that despite an increase in the prevalence of individual mutations, the IPT-SP coupled with the EPI has no major impact on DHFR and DHPS combined mutations.
The capacity of ten molecules for reversing resistance in Plasmodium falciparum in vitro to quino... more The capacity of ten molecules for reversing resistance in Plasmodium falciparum in vitro to quinoline antimalarial drugs, such as chloroquine (CQ), quinine (QN), mefloquine (MQ) and monodesethylamodiaquine (MDAQ), was assessed against 27 Plasmodium falciparum isolates. Four of these compounds were 9,10-dihydroethanoanthracene derivatives (DEAs). These DEAs reversed 75 to 92% of the CQ resistant strains. These synthetic compounds were more effective in combination with CQ than verapamil, ketotifen, chlorpromazine, reserpine or nicardipine, which reversed less than 50% of the CQ resistant strains. DEAs significantly reversed 67 to 100% of MDAQ resistant parasites. These compounds were more effective in combination with MDAQ than ketotifen (60% of reversal), chlorpromazine (45%), verapamil (33%), reserpine (30%) or nicardipine (9%). The reversal activity of MQ resistance was less pronounced, regardless of the molecule tested, and was homogeneous with a rate ranging from 42% for ketotifen to 58% for reserpine, nicardipine, verapamil and cyproheptadine. The four DEAs significantly reversed 50 to 55% of the parasites resistant to MQ. Fifty-six to 78 % of the QN resistant parasites were reversed by the synthetic DEAs. There were few differences in the rate of reversal activity on QN resistant strains between the ten compounds, with rates ranging between 56 to 78% for the ten chemosensitizers. The use of DEAs in combination with quinoline seems to be thus a promising strategy for limiting the development of drug resistant strains and for treating patients in drug resistant areas.
Background Although the World Health Organization recommends replacing quinine (QN) by artesunate... more 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 Pla...
Background One of the major complications of Plasmodium falciparum infection is cerebral malaria ... more Background One of the major complications of Plasmodium falciparum infection is cerebral malaria (CM), which causes one million deaths worldwide each year, results in long-term neurological sequelae and the treatment for which is only partially effective. Statins are recognized to have an immunomodulatory action, attenuate sepsis and have a neuroprotective effect. Atorvastatin (AVA) has shown in vitro anti-malarial activity and has improved the activity of mefloquine (MQ) and quinine. Methods The efficiency of 40 mg/kg intraperitoneal AVA, alone or in association with MQ, was assessed in an experimental Plasmodium berghei ANKA rodent parasite model of CM and performed according to different therapeutic schemes. The effects on experimental CM were assessed through the evaluation of brain histopathological changes and neuronal apoptosis by TUNEL staining. Results AVA alone in the therapeutic scheme show no effect on survival, but the prophylactic scheme employing AVA associated with M...
Background: The aim of the present work was to assess the in vitro cross-resistance of pyronaridi... more Background: The aim of the present work was to assess the in vitro cross-resistance of pyronaridine with other quinoline drugs, artesunate and several other commonly used anti-malarials and to evaluate whether decreased susceptibility to pyronaridine could be associated with genetic polymorphisms in genes involved in reduced quinoline susceptibility, such as pfcrt, pfmdr1, pfmrp and pfnhe. Methods: The in vitro chemosusceptibility profiles of 23 strains of Plasmodium falciparum were analysed by the standard 42-hour 3 H-hypoxanthine uptake inhibition method for pyronaridine, artesunate, chloroquine, monodesethylamodiaquine, quinine, mefloquine, lumefantrine, atovaquone, pyrimethamine and doxycycline. Genotypes were assessed for pfcrt, pfmdr1, pfnhe-1 and pfmrp genes. Results: The IC 50 values for pyronaridine ranged from 15 to 49 nM (geometric mean = 23.1 nM). A significant positive correlation was found between responses to pyronaridine and responses to artesunate (r 2 = 0.20; P = 0.0317) but too low to suggest cross-resistance. No significant correlation was found between pyronaridine IC 50 and responses to other anti-malarials. Significant associations were not found between pyronaridine IC 50 and polymorphisms in pfcrt, pfmdr1, pfmrp or pfnhe-1. Conclusion: There was an absence of cross-resistance between pyronaridine and quinolines, and the IC 50 values for pyronaridine were found to be unrelated to mutations in the transport protein genes pfcrt, pfmdr1, pfmrp or pfnhe-1, known to be involved in quinoline resistance. These results confirm the interest and the efficacy of the use of a combination of pyronaridine and artesunate in areas in which parasites are resistant to quinolines.
Background In 2006, the Senegalese National Malaria Control Programme recommended artemisinin-bas... more 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 amo...
Background: Recently, Plasmodium falciparum parasites bearing Pfdhfr I164L single mutation were f... more Background: Recently, Plasmodium falciparum parasites bearing Pfdhfr I164L single mutation were found in Madagascar. These new mutants may challenge the use of antifolates for the intermittent preventive treatment of malaria during pregnancy (IPTp). Assays with transgenic bacteria suggested that I164L parasites have a wild-type phenotype for pyrimethamine but it had to be confirmed by testing the parasites themselves. Methods: Thirty Plasmodium falciparum clinical isolates were collected in 2008 in the southeast of Madagascar. A part of Pfdhfr gene encompassing codons 6 to 206 was amplified by PCR and the determination of the presence of single nucleotide polymorphisms was performed by DNA sequencing. The multiplicity of infection was estimated by using an allelic family-specific nested PCR. Isolates that appeared monoclonal were submitted to culture adaptation. Determination of IC 50s to pyrimethamine was performed on adapted isolates. Results: Four different Pfdhfr alleles were found: the 164L single mutant-type (N = 13), the wild-type (N = 7), the triple mutant-type 51I/59R/108N (N = 9) and the double mutant-type 108N/164L (N = 1). Eleven out 30 (36.7%) of P. falciparum isolates were considered as monoclonal infection. Among them, five isolates were successfully adapted in culture and tested for pyrimethamine in vitro susceptibility. The wild-type allele was the most susceptible with a 50% inhibitory concentration (IC 50) < 10 nM. The geometric mean of IC 50 of the three I164L mutant isolates was 6-fold higher than the wild-type with 61.3 nM (SD = 3.2 nM, CI95%: 53.9-69.7 nM). These values remained largely below the IC 50 of the triple mutant parasite (13,804 nM). Conclusion: The IC 50 s of the I164L mutant isolates were significantly higher than those of the wild-type (6-fold higher) and close from those usually reported for simple mutants S108N (roughly10-fold higher than wild type). Given the observed values, the determination of IC 50 s directly on parasites did not confirm what has been found on transgenic bacteria. The prevalence increase of the Pfdhfr I164L single mutant parasite since 2006 could be explained by the selective advantage of this allele under sulphadoxine-pyrimethamine pressure. The emergence of highly resistant alleles should be considered in the future, in particular because an unexpected double mutant-type allele S108N/I164L has been already detected.
We determined the level of in vitro resistance of Plasmodium falciparum parasites to standard ant... more We determined the level of in vitro resistance of Plasmodium falciparum parasites to standard antimalarial drugs, such as chloroquine, quinine, amodiaquine, halofantrine, mefloquine, cycloguanil, and pyrimethamine, and to new compounds, such as dihydroartemisinin, doxycycline, atovaquone, and lumefantrine. The in vitro resistance to chloroquine reached 75.5%. Twenty-eight percent of the isolates were intermediate or had reduced susceptibility to quinine. Seventy-six percent and 96% of the tested isolates showed in vitro resistance or intermediate susceptibilities to cycloguanil and pyrimethamine, respectively. Only 2% of the parasites demonstrated in vitro resistance to monodesethylamodiaquine. No resistance was shown with halofantrine, lumefantrine, dihydroartemisinin, or atovaquone. Halofantrine, mefloquine, and lumefantrine demonstrated high correlation. No cross-resistance was identified between responses to monodesethyl-amodiaquine, dihydroartemisinin, atovaquone, and cycloguanil. Since the level of chloroquine resistance in vitro exceed an unacceptable upper limit, high rates of in vitro resistance to pyrimethamine and cycloguanil and diminution of the susceptibility to quinine, antimalarial drugs used in combination, such as amodiaquine, artemisinin derivatives, mefloquine, lumefantrine, or atovaquone, seem to be appropriate alternatives for the first line of treatment of acute, uncomplicated P. falciparum malaria.
The aim of the study was to assess the in vitro potentiating effects of atorvastatin, a 3-hydroxy... more The aim of the study was to assess the in vitro potentiating effects of atorvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, in combination with mefloquine, chloroquine or monodesethylamodiaquine against Plasmodium falciparum and to evaluate whether the effects of atorvastatin could be associated with mutations or gene copy number in multidrug resistance (MDR)-like protein genes. Methods: The susceptibilities of 21 parasite strains to combinations of atorvastatin with mefloquine, chloroquine or monodesethylamodiaquine were assessed using the in vitro isotopic microtest. Genotypes and gene copy number were assessed for pfmdr1, pfmdr2 and pfmrp genes. Results: Atorvastatin demonstrated synergistic effects in combination with mefloquine. The mefloquine IC 50 (50% inhibitory concentration) was reduced by 7%, 24% and 37% in the presence of atorvastatin at concentrations of 0.1, 0.5 and 1.0 mM, respectively. The synergistic effect of atorvastatin on the response to mefloquine was significantly associated with pfmdr1 copy number. The concentration of atorvastatin that could reduce the IC 50 of mefloquine by 50% was 2.4+1.3 mM for the 12 strains that contained one copy of pfmdr1 and 5.8+2.1 mM for the 9 strains that contained two copies or more. The synergistic effect of atorvastatin in combination with mefloquine was found to be significantly unrelated to mutations in pfmdr1, pfmdr2 or pfmrp genes. Conclusions: The synergy of the effect of mefloquine at concentrations relevant to its achievable plasma concentrations in patients taking 80 mg of atorvastatin daily suggests that atorvastatin will be a good candidate in combination with mefloquine for malaria treatment.
To the Editor: Amplification and overexpression of the Plasmodium falciparum multidrug resistance... more To the Editor: Amplification and overexpression of the Plasmodium falciparum multidrug resistance 1 gene (Pfmdr1) have been associated with mefloquine resistance in P. falciparum malaria in Asia (1). Amplification of Pfmdr1 in Africa has occurred rarely. Only 12 isolates with >2 copies of Pfmdr1 were identified in Africa during 1993-2012: 3 in Côte d'Ivoire (2,3), 1 in Burkina Faso (3), 1 in Togo (3), 3 in eastern Sudan (4), 2 in Kenya (5,6), and 1 Senegal (7). Another isolate was obtained in a patient from Benin who did not respond clinically to mefloquine treatment (8). Pfmdr1 amplification has not been found in samples collected either before or after treatment for recurring P. falciparum infection in Africa in many studies. In Dakar, Senegal, and its surrounding suburbs, malaria is transmitted with spatial heterogeneity to the human mosquito bite rate, which ranged from 0.1 to 250 bites per person per night during the rainy seasons of 2007-2010. P. falciparum isolates from patients with malaria who lived in Dakar (>80%) and the surrounding area and did not travel during the previous month were obtained during the rainy seasons of October 2009-January 2010 (172 patients, 42% female) and August 2010-January 2011 (129 patients, 38% female). Informed verbal consent from the patients and/or their parents/guardians was obtained before blood collection; the study was approved by the ethical committee of the Hôpital Principal de Dakar. Of the 301 patients, 54% were recruited from the emergency department during each of the 2 seasons;
We have analyzed the profiles of 23 of Plasmodium falciparum strains for their in vitro chemosusc... more We have analyzed the profiles of 23 of Plasmodium falciparum strains for their in vitro chemosusceptibilities to piperaquine (PPQ), dihydroartemisinin (DHA), chloroquine, monodesethylamodiaquine, quinine, mefloquine, lumefantrine, atovaquone, pyrimethamine, and doxycycline (DOX) in association with polymorphisms in genes involved in quinoline resistance (Plasmodium falciparum crt [pfcrt], pfmdr1, pfmrp, and pfnhe). The 50% inhibitory concentrations (IC50s) for PPQ ranged from 29 to 98 nM (geometric mean = 57.8 nM, 95% confidence interval [CI] = 51 to 65) and from 0.4 to 5.8 nM for DHA (geometric mean = 1.8 nM, 95% CI = 1.4 to 2.3). We found a significant positive correlation between the responses to PPQ and DHA (r 2 = 0.17; P = 0.0495) and between the responses to PPQ and DOX (r 2 = 0.41; P = 0.001). We did not find a significant association between the PPQ IC50 (0.0525 < P < 0.9247) or the DHA IC50 (0.0138 < P < 0.9018) and polymorphisms in the pfcrt, pfmdr1, pfmrp, and...
The in vitro activities of cyclines (tetracycline, doxycycline, minocycline, oxytetracycline, and... more The in vitro activities of cyclines (tetracycline, doxycycline, minocycline, oxytetracycline, and rolitetracycline), macrolides (erythromycin, spiramycin, roxithromycin, and lincomycin), quinolones (norfloxacin and ofloxacin), rifampin, thiamphenicol, tobramycin, metronidazole, vancomycin, phosphomycin, and cephalosporins (cephalexin, cefaclor, cefamandole, cefuroxime, ceftriazone, cefotaxime, and cefoxitin) were evaluated on Plasmodium falciparum clones, using an isotopic, micro-drug susceptibility test. Only tetracyclines, macrolides, quinolones, and rifampin demonstrated in vitro activity against P. falciparum , which increased after a prolonged exposure (96 or 144 h). In the presence of iron (FeCl 3 ), only the activities of tetracyclines and norfloxacin were decreased. Their in vitro activity against intraerythrocytic stages of multidrug-resistant P. falciparum and their efficacy in vivo favor the use of antibiotics as antimalarial drugs. However, due to their slow antimalarial...
Atorvastatin (AVA) is a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor. AVA exposure r... more Atorvastatin (AVA) is a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor. AVA exposure resulted in the reduced in vitro growth of 22 Plasmodium falciparum strains, with the 50% inhibitory concentrations (IC 50 s) ranging from 2.5 μM to 10.8 μM. A significant positive correlation was found between the strains’ responses to AVA and mefloquine ( r = 0.553; P = 0.008). We found no correlation between the responses to AVA and to chloroquine, quinine, monodesethylamodiaquine, lumefantrine, dihydroartemisinin, atovaquone, or doxycycline. These data could suggest that the mechanism of AVA uptake and/or the mode of action of AVA is different from those for other antimalarial drugs. The IC 50 s for AVA were unrelated to the occurrence of mutations in the transport protein genes involved in quinoline antimalarial drug resistance, such as the P. falciparum crt , mdr1 , mrp , and nhe-1 genes. Therefore, AVA can be ruled out as a substrate for the transport proteins (CRT, Pgh1, and MRP) ...
We have analyzed the profiles of 23 of Plasmodium falciparum strains for their in vitro chemosusc... more We have analyzed the profiles of 23 of Plasmodium falciparum strains for their in vitro chemosusceptibilities to piperaquine (PPQ), dihydroartemisinin (DHA), chloroquine, monodesethylamodiaquine, quinine, mefloquine, lumefantrine, atovaquone, pyrimethamine, and doxycycline (DOX) in association with polymorphisms in genes involved in quinoline resistance (Plasmodium falciparum crt [pfcrt], pfmdr1, pfmrp, and pfnhe). The 50% inhibitory concentrations (IC 50 s) for PPQ ranged from 29 to 98 nM (geometric mean ؍ 57.8 nM, 95% confidence interval [CI] ؍ 51 to 65) and from 0.4 to 5.8 nM for DHA (geometric mean ؍ 1.8 nM, 95% CI ؍ 1.4 to 2.3). We found a significant positive correlation between the responses to PPQ and DHA (r 2 ؍ 0.17; P ؍ 0.0495) and between the responses to PPQ and DOX (r 2 ؍ 0.41; P ؍ 0.001). We did not find a significant association between the PPQ IC 50 (0.0525 < P < 0.9247) or the DHA IC 50 (0.0138 < P < 0.9018) and polymorphisms in the pfcrt, pfmdr1, pfmrp, and pfnhe-1 genes. There was an absence of cross-resistance with quinolines, and the IC 50 s for PPQ and DHA were found to be unrelated to mutations in the pfcrt, pfmdr1, pfmrp, and pfnhe-1 transport protein genes, which are involved in quinoline antimalarial drug resistance. These results confirm the interest in and the efficacy of the combination of PPQ and DHA for areas in which parasites are resistant to chloroquine or other quinolines.
There is an urgent need for the discovery of new anti-malarial drugs and combination therapy. A c... more There is an urgent need for the discovery of new anti-malarial drugs and combination therapy. A combinatorial approach protects each drug from the development of resistance and reduces generally the overall transmission rate of malaria. Statins, the inhibitors of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase and a family of lipid-lowering drugs, have in vitro anti-malarial properties, and more specially atorvastatin. However, atorvastatin has a short elimination half-life (14 hours) and an efficient combination of anti-malarial drugs must associate a drug with a short elimination half-life and a drug with a long elimination half-life. The objective of the present work was to identify new potential partners among standard new anti-malarial drugs with long elimination half-life, such as lumefantrine, piperaquine, pyronaridine and atovaquone, to improve the in vitro activity of atorvastatin against different Plasmodium falciparum strains to treat uncomplicated malaria. In vitro inter...
The aim of this study was to evaluate the cultivation system in which the proper atmospheric cond... more The aim of this study was to evaluate the cultivation system in which the proper atmospheric conditions for growing Plasmodium falciparum parasites were maintained in a sealed bag. The Genbag® system associated with the atmospheric generators for capnophilic bacteria Genbag CO2® was used for in vitro susceptibility test of nine standard anti-malarial drugs and compared to standard incubator conditions. The susceptibility of 36 pre-identified parasite strains from a wide panel of countries was assessed for nine standard anti-malarial drugs (chloroquine, quinine, mefloquine, monodesethylamodiaquine, lumefantrine, dihydroartemisinin, atovaquone and pyrimethamine) by the standard 42-hour 3H-hypoxanthine uptake inhibition method using the Genbag CO2® system and compared to controlled incubator conditions (5% CO2 and 10% O2). The counts per minute values in the control wells in incubator atmospheric conditions (5% CO2 and 10% O2) were significantly higher than those of Genbag® conditions ...
ABSTRACTThe involvement ofPfmdr1(Plasmodium falciparummultidrug resistance 1) polymorphisms in an... more ABSTRACTThe involvement ofPfmdr1(Plasmodium falciparummultidrug resistance 1) polymorphisms in antimalarial drug resistance is still debated. Here, we evaluate the association between polymorphisms inPfmdr1(N86Y, Y184F, S1034C, N1042D, and D1246Y) andPfcrt(K76T) andin vitroresponses to chloroquine (CQ), mefloquine (MQ), lumefantrine (LMF), quinine (QN), monodesethylamodiaquine (MDAQ), and dihydroartemisinin (DHA) in 174Plasmodium falciparumisolates from Dakar, Senegal. ThePfmdr186Y 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. ThePfmdr186Y 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. ThePfmdr1184F mutation was not associated with various susceptibility responses to the 6 antimalarial drug...
Several studies have shown the efficacy of the intermittent preventive treatment (IPT) using sulf... more Several studies have shown the efficacy of the intermittent preventive treatment (IPT) using sulfadoxine-pyrimethamine (SP) coupled with the expanded program of immunization (EPI) in infants. However, its adoption as a strategy is conditioned by the long-term efficacy of SP. The impact of IPT-SP coupled with the EPI on the prevalence of markers of resistance to SP was evaluated during this study conducted in Southern Senegal. Three cross-sectional surveys in two health districts (IPT+) were conducted prior to the implementation, 1 year, and 2 years after. A third district located between the two districts served as a test zone (IPT-). PCR tests were carried out from filter papers collected in children under five for the two first measures and from positive rapid diagnostic tests in the same population for the third measure. Mutations in codons 51, 59, and 108 of the DHFR gene and in codons 437 and 540 of the DHPS were analyzed. The results showed that the prevalence of DHFR triple mutation was more frequent after 2 years in IPT+ areas. Regarding quadruple mutation, DHFR (51, 59, and 108) and DHPS (437), no difference was noted between the two areas. The quintuple mutation was not observed after 2 years of implementation in both areas. However, an individual analysis showed significant differences in the individual mutation points 51, 59, 108, and 437. This study reveals that despite an increase in the prevalence of individual mutations, the IPT-SP coupled with the EPI has no major impact on DHFR and DHPS combined mutations.
The capacity of ten molecules for reversing resistance in Plasmodium falciparum in vitro to quino... more The capacity of ten molecules for reversing resistance in Plasmodium falciparum in vitro to quinoline antimalarial drugs, such as chloroquine (CQ), quinine (QN), mefloquine (MQ) and monodesethylamodiaquine (MDAQ), was assessed against 27 Plasmodium falciparum isolates. Four of these compounds were 9,10-dihydroethanoanthracene derivatives (DEAs). These DEAs reversed 75 to 92% of the CQ resistant strains. These synthetic compounds were more effective in combination with CQ than verapamil, ketotifen, chlorpromazine, reserpine or nicardipine, which reversed less than 50% of the CQ resistant strains. DEAs significantly reversed 67 to 100% of MDAQ resistant parasites. These compounds were more effective in combination with MDAQ than ketotifen (60% of reversal), chlorpromazine (45%), verapamil (33%), reserpine (30%) or nicardipine (9%). The reversal activity of MQ resistance was less pronounced, regardless of the molecule tested, and was homogeneous with a rate ranging from 42% for ketotifen to 58% for reserpine, nicardipine, verapamil and cyproheptadine. The four DEAs significantly reversed 50 to 55% of the parasites resistant to MQ. Fifty-six to 78 % of the QN resistant parasites were reversed by the synthetic DEAs. There were few differences in the rate of reversal activity on QN resistant strains between the ten compounds, with rates ranging between 56 to 78% for the ten chemosensitizers. The use of DEAs in combination with quinoline seems to be thus a promising strategy for limiting the development of drug resistant strains and for treating patients in drug resistant areas.
Background Although the World Health Organization recommends replacing quinine (QN) by artesunate... more 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 Pla...
Background One of the major complications of Plasmodium falciparum infection is cerebral malaria ... more Background One of the major complications of Plasmodium falciparum infection is cerebral malaria (CM), which causes one million deaths worldwide each year, results in long-term neurological sequelae and the treatment for which is only partially effective. Statins are recognized to have an immunomodulatory action, attenuate sepsis and have a neuroprotective effect. Atorvastatin (AVA) has shown in vitro anti-malarial activity and has improved the activity of mefloquine (MQ) and quinine. Methods The efficiency of 40 mg/kg intraperitoneal AVA, alone or in association with MQ, was assessed in an experimental Plasmodium berghei ANKA rodent parasite model of CM and performed according to different therapeutic schemes. The effects on experimental CM were assessed through the evaluation of brain histopathological changes and neuronal apoptosis by TUNEL staining. Results AVA alone in the therapeutic scheme show no effect on survival, but the prophylactic scheme employing AVA associated with M...
Background: The aim of the present work was to assess the in vitro cross-resistance of pyronaridi... more Background: The aim of the present work was to assess the in vitro cross-resistance of pyronaridine with other quinoline drugs, artesunate and several other commonly used anti-malarials and to evaluate whether decreased susceptibility to pyronaridine could be associated with genetic polymorphisms in genes involved in reduced quinoline susceptibility, such as pfcrt, pfmdr1, pfmrp and pfnhe. Methods: The in vitro chemosusceptibility profiles of 23 strains of Plasmodium falciparum were analysed by the standard 42-hour 3 H-hypoxanthine uptake inhibition method for pyronaridine, artesunate, chloroquine, monodesethylamodiaquine, quinine, mefloquine, lumefantrine, atovaquone, pyrimethamine and doxycycline. Genotypes were assessed for pfcrt, pfmdr1, pfnhe-1 and pfmrp genes. Results: The IC 50 values for pyronaridine ranged from 15 to 49 nM (geometric mean = 23.1 nM). A significant positive correlation was found between responses to pyronaridine and responses to artesunate (r 2 = 0.20; P = 0.0317) but too low to suggest cross-resistance. No significant correlation was found between pyronaridine IC 50 and responses to other anti-malarials. Significant associations were not found between pyronaridine IC 50 and polymorphisms in pfcrt, pfmdr1, pfmrp or pfnhe-1. Conclusion: There was an absence of cross-resistance between pyronaridine and quinolines, and the IC 50 values for pyronaridine were found to be unrelated to mutations in the transport protein genes pfcrt, pfmdr1, pfmrp or pfnhe-1, known to be involved in quinoline resistance. These results confirm the interest and the efficacy of the use of a combination of pyronaridine and artesunate in areas in which parasites are resistant to quinolines.
Background In 2006, the Senegalese National Malaria Control Programme recommended artemisinin-bas... more 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 amo...
Background: Recently, Plasmodium falciparum parasites bearing Pfdhfr I164L single mutation were f... more Background: Recently, Plasmodium falciparum parasites bearing Pfdhfr I164L single mutation were found in Madagascar. These new mutants may challenge the use of antifolates for the intermittent preventive treatment of malaria during pregnancy (IPTp). Assays with transgenic bacteria suggested that I164L parasites have a wild-type phenotype for pyrimethamine but it had to be confirmed by testing the parasites themselves. Methods: Thirty Plasmodium falciparum clinical isolates were collected in 2008 in the southeast of Madagascar. A part of Pfdhfr gene encompassing codons 6 to 206 was amplified by PCR and the determination of the presence of single nucleotide polymorphisms was performed by DNA sequencing. The multiplicity of infection was estimated by using an allelic family-specific nested PCR. Isolates that appeared monoclonal were submitted to culture adaptation. Determination of IC 50s to pyrimethamine was performed on adapted isolates. Results: Four different Pfdhfr alleles were found: the 164L single mutant-type (N = 13), the wild-type (N = 7), the triple mutant-type 51I/59R/108N (N = 9) and the double mutant-type 108N/164L (N = 1). Eleven out 30 (36.7%) of P. falciparum isolates were considered as monoclonal infection. Among them, five isolates were successfully adapted in culture and tested for pyrimethamine in vitro susceptibility. The wild-type allele was the most susceptible with a 50% inhibitory concentration (IC 50) < 10 nM. The geometric mean of IC 50 of the three I164L mutant isolates was 6-fold higher than the wild-type with 61.3 nM (SD = 3.2 nM, CI95%: 53.9-69.7 nM). These values remained largely below the IC 50 of the triple mutant parasite (13,804 nM). Conclusion: The IC 50 s of the I164L mutant isolates were significantly higher than those of the wild-type (6-fold higher) and close from those usually reported for simple mutants S108N (roughly10-fold higher than wild type). Given the observed values, the determination of IC 50 s directly on parasites did not confirm what has been found on transgenic bacteria. The prevalence increase of the Pfdhfr I164L single mutant parasite since 2006 could be explained by the selective advantage of this allele under sulphadoxine-pyrimethamine pressure. The emergence of highly resistant alleles should be considered in the future, in particular because an unexpected double mutant-type allele S108N/I164L has been already detected.
We determined the level of in vitro resistance of Plasmodium falciparum parasites to standard ant... more We determined the level of in vitro resistance of Plasmodium falciparum parasites to standard antimalarial drugs, such as chloroquine, quinine, amodiaquine, halofantrine, mefloquine, cycloguanil, and pyrimethamine, and to new compounds, such as dihydroartemisinin, doxycycline, atovaquone, and lumefantrine. The in vitro resistance to chloroquine reached 75.5%. Twenty-eight percent of the isolates were intermediate or had reduced susceptibility to quinine. Seventy-six percent and 96% of the tested isolates showed in vitro resistance or intermediate susceptibilities to cycloguanil and pyrimethamine, respectively. Only 2% of the parasites demonstrated in vitro resistance to monodesethylamodiaquine. No resistance was shown with halofantrine, lumefantrine, dihydroartemisinin, or atovaquone. Halofantrine, mefloquine, and lumefantrine demonstrated high correlation. No cross-resistance was identified between responses to monodesethyl-amodiaquine, dihydroartemisinin, atovaquone, and cycloguanil. Since the level of chloroquine resistance in vitro exceed an unacceptable upper limit, high rates of in vitro resistance to pyrimethamine and cycloguanil and diminution of the susceptibility to quinine, antimalarial drugs used in combination, such as amodiaquine, artemisinin derivatives, mefloquine, lumefantrine, or atovaquone, seem to be appropriate alternatives for the first line of treatment of acute, uncomplicated P. falciparum malaria.
The aim of the study was to assess the in vitro potentiating effects of atorvastatin, a 3-hydroxy... more The aim of the study was to assess the in vitro potentiating effects of atorvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, in combination with mefloquine, chloroquine or monodesethylamodiaquine against Plasmodium falciparum and to evaluate whether the effects of atorvastatin could be associated with mutations or gene copy number in multidrug resistance (MDR)-like protein genes. Methods: The susceptibilities of 21 parasite strains to combinations of atorvastatin with mefloquine, chloroquine or monodesethylamodiaquine were assessed using the in vitro isotopic microtest. Genotypes and gene copy number were assessed for pfmdr1, pfmdr2 and pfmrp genes. Results: Atorvastatin demonstrated synergistic effects in combination with mefloquine. The mefloquine IC 50 (50% inhibitory concentration) was reduced by 7%, 24% and 37% in the presence of atorvastatin at concentrations of 0.1, 0.5 and 1.0 mM, respectively. The synergistic effect of atorvastatin on the response to mefloquine was significantly associated with pfmdr1 copy number. The concentration of atorvastatin that could reduce the IC 50 of mefloquine by 50% was 2.4+1.3 mM for the 12 strains that contained one copy of pfmdr1 and 5.8+2.1 mM for the 9 strains that contained two copies or more. The synergistic effect of atorvastatin in combination with mefloquine was found to be significantly unrelated to mutations in pfmdr1, pfmdr2 or pfmrp genes. Conclusions: The synergy of the effect of mefloquine at concentrations relevant to its achievable plasma concentrations in patients taking 80 mg of atorvastatin daily suggests that atorvastatin will be a good candidate in combination with mefloquine for malaria treatment.
To the Editor: Amplification and overexpression of the Plasmodium falciparum multidrug resistance... more To the Editor: Amplification and overexpression of the Plasmodium falciparum multidrug resistance 1 gene (Pfmdr1) have been associated with mefloquine resistance in P. falciparum malaria in Asia (1). Amplification of Pfmdr1 in Africa has occurred rarely. Only 12 isolates with >2 copies of Pfmdr1 were identified in Africa during 1993-2012: 3 in Côte d'Ivoire (2,3), 1 in Burkina Faso (3), 1 in Togo (3), 3 in eastern Sudan (4), 2 in Kenya (5,6), and 1 Senegal (7). Another isolate was obtained in a patient from Benin who did not respond clinically to mefloquine treatment (8). Pfmdr1 amplification has not been found in samples collected either before or after treatment for recurring P. falciparum infection in Africa in many studies. In Dakar, Senegal, and its surrounding suburbs, malaria is transmitted with spatial heterogeneity to the human mosquito bite rate, which ranged from 0.1 to 250 bites per person per night during the rainy seasons of 2007-2010. P. falciparum isolates from patients with malaria who lived in Dakar (>80%) and the surrounding area and did not travel during the previous month were obtained during the rainy seasons of October 2009-January 2010 (172 patients, 42% female) and August 2010-January 2011 (129 patients, 38% female). Informed verbal consent from the patients and/or their parents/guardians was obtained before blood collection; the study was approved by the ethical committee of the Hôpital Principal de Dakar. Of the 301 patients, 54% were recruited from the emergency department during each of the 2 seasons;
We have analyzed the profiles of 23 of Plasmodium falciparum strains for their in vitro chemosusc... more We have analyzed the profiles of 23 of Plasmodium falciparum strains for their in vitro chemosusceptibilities to piperaquine (PPQ), dihydroartemisinin (DHA), chloroquine, monodesethylamodiaquine, quinine, mefloquine, lumefantrine, atovaquone, pyrimethamine, and doxycycline (DOX) in association with polymorphisms in genes involved in quinoline resistance (Plasmodium falciparum crt [pfcrt], pfmdr1, pfmrp, and pfnhe). The 50% inhibitory concentrations (IC50s) for PPQ ranged from 29 to 98 nM (geometric mean = 57.8 nM, 95% confidence interval [CI] = 51 to 65) and from 0.4 to 5.8 nM for DHA (geometric mean = 1.8 nM, 95% CI = 1.4 to 2.3). We found a significant positive correlation between the responses to PPQ and DHA (r 2 = 0.17; P = 0.0495) and between the responses to PPQ and DOX (r 2 = 0.41; P = 0.001). We did not find a significant association between the PPQ IC50 (0.0525 < P < 0.9247) or the DHA IC50 (0.0138 < P < 0.9018) and polymorphisms in the pfcrt, pfmdr1, pfmrp, and...
The in vitro activities of cyclines (tetracycline, doxycycline, minocycline, oxytetracycline, and... more The in vitro activities of cyclines (tetracycline, doxycycline, minocycline, oxytetracycline, and rolitetracycline), macrolides (erythromycin, spiramycin, roxithromycin, and lincomycin), quinolones (norfloxacin and ofloxacin), rifampin, thiamphenicol, tobramycin, metronidazole, vancomycin, phosphomycin, and cephalosporins (cephalexin, cefaclor, cefamandole, cefuroxime, ceftriazone, cefotaxime, and cefoxitin) were evaluated on Plasmodium falciparum clones, using an isotopic, micro-drug susceptibility test. Only tetracyclines, macrolides, quinolones, and rifampin demonstrated in vitro activity against P. falciparum , which increased after a prolonged exposure (96 or 144 h). In the presence of iron (FeCl 3 ), only the activities of tetracyclines and norfloxacin were decreased. Their in vitro activity against intraerythrocytic stages of multidrug-resistant P. falciparum and their efficacy in vivo favor the use of antibiotics as antimalarial drugs. However, due to their slow antimalarial...
Atorvastatin (AVA) is a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor. AVA exposure r... more Atorvastatin (AVA) is a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor. AVA exposure resulted in the reduced in vitro growth of 22 Plasmodium falciparum strains, with the 50% inhibitory concentrations (IC 50 s) ranging from 2.5 μM to 10.8 μM. A significant positive correlation was found between the strains’ responses to AVA and mefloquine ( r = 0.553; P = 0.008). We found no correlation between the responses to AVA and to chloroquine, quinine, monodesethylamodiaquine, lumefantrine, dihydroartemisinin, atovaquone, or doxycycline. These data could suggest that the mechanism of AVA uptake and/or the mode of action of AVA is different from those for other antimalarial drugs. The IC 50 s for AVA were unrelated to the occurrence of mutations in the transport protein genes involved in quinoline antimalarial drug resistance, such as the P. falciparum crt , mdr1 , mrp , and nhe-1 genes. Therefore, AVA can be ruled out as a substrate for the transport proteins (CRT, Pgh1, and MRP) ...
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Papers by Eric Baret