Eric Baret

Plasmodium falciparum Na+/H+ Exchanger 1 Transporter Is Involved in Reduced Susceptibility to Quinine

ABSTRACT Polymorphisms in the Plasmodium falciparum crt (Pfcrt), Pfmdr1, and Pfmrp genes were not significantly associated with quinine (QN) 50% inhibitory concentrations (IC50s) in 23 strains of Plasmodium falciparum. An increased number of DNNND repeats in Pfnhe-1 microsatellite ms4760 was associated with an increased IC50 of QN (P = 0.0007). Strains with only one DNNND repeat were more susceptible to QN (mean IC50 of 154 nM). Strains with two DNNND repeats had intermediate susceptibility to QN (mean IC50 of 548 nM). Strains with three DNNND repeats had reduced susceptibility to QN (mean IC50 of 764 nM). Increased numbers of NHNDNHNNDDD repeats were associated with a decreased IC50 of QN (P = 0.0020). Strains with profile 7 for Pfnhe-1 ms4760 (ms4760-7) were significantly associated with reduced QN susceptibility (mean IC50 of 764 nM). The determination of DNNND and NHNDNHNNDDD repeats in Pfnhe-1 ms4760 could be a good marker of QN resistance and provide an attractive surveillance method to monitor temporal trends in P. falciparum susceptibility to QN. The validity of the markers should be further supported by analyzing more isolates.

Prevalence of In Vitro Resistance to Eleven Standard or New Antimalarial Drugs among Plasmodium falciparum Isolates from Pointe-Noire, Republic of the Congo

ABSTRACT 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.

In Vitro Activity of Ferroquine Is Independent of Polymorphisms in Transport Protein Genes Implicated in Quinoline Resistance in Plasmodium falciparum

ABSTRACT The in vitro activity of ferroquine (FQ) (SR97193), a 4-aminoquinoline antimalarial compound that contains a ferrocenic nucleus, against 15 Plasmodium falciparum strains was assessed and compared with those of chloroquine (CQ), quinine (QN), monodesethylamodiaquine (MDAQ), and mefloquine (MQ). These 15 strains were genotyped for polymorphisms in quinoline resistance-associated genes such as Pfcrt, Pfmdr1, Pfmrp, and Pfnhe-1. FQ was highly active against CQ-resistant parasites or in parasites with reduced susceptibility to QN, MDAQ, or MQ. Encouragingly, we did not find a correlation between responses to FQ and those to other quinoline drugs. These results suggest that no cross-resistance exits between FQ and CQ or quinoline antimalarial drugs. Mutations in codons 74, 75, 76, 220, 271, 326, 356, and 371 of the Pfcrt gene; codons 86, 184, 1034, 1042, and 1246 of the Pfmdr1 gene; and codons 191 and 437 of the Pfmrp gene were not significantly associated with P. falciparum susceptibility to FQ. Neither the number of ms4760 DNNND or DDNHNDNHNN repeats in Pfnhe-1 nor the profile of ms4760 was significantly associated with the FQ in vitro response. These data suggest the FQ may not interact with transport proteins in quinoline-resistant parasites. The present results justify further clinical trials of FQ in multidrug resistance areas.

Atorvastatin Is 10-Fold More Active In Vitro than Other Statins against Plasmodium falciparum

Statins, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, influence a broad array of pathogenic microorganisms. Lovastatin reduces the intracellular growth of Salmonella enterica serovar Typhimurium in cultured macrophages, while atorvastatin does the same in a mouse model (2). Lovastatin additionally reduces the growth of Candida albicans by inhibiting the sterol pathway (11). Statins interfere severely with the growth of protozoan parasites of the Trypanosomatidae family such as Trypanosoma cruzi and various Leishmania species (8, 12, 13). HMG-CoA reductase has been detected in Trypanosoma and Leishmania (3, 8). The presence of an HMG-CoA homolog was not revealed by BLASTX analysis of the Plasmodium falciparum sequence with other protozoal HMG-CoA protein sequences. However, as reported previously, treatment in vitro of Plasmodium falciparum with 120 or 240 μM mevastatin inhibited parasite growth (4, 9). The susceptibilities to simvastatin, simvastatin sodium salt, pravastatin sodium salt, lovastatin, fluvastatin sodium salt, mevastatin, mevastatin sodium salt (Calbiochem, Merck, Germany), and atorvastatin calcium salt (Molekula, United Kingdom) were assessed in vitro against chloroquine-susceptible P. falciparum strains 3D7 (Africa), D6 (Sierra Leone), and IMT031 (Gabon) and chloroquine-resistant strains W2 (Indochina), Bre1 (Brazil), and FCR3 (The Gambia). Lovastatin and mevastatin were converted to the active form by dissolving the lactone form in 100 μl of 100% ethanol, adding 200 μl of 0.2 M KOH, and then adding 0.2 M HCl for neutralization to pH 7.2 (5). Simvastatin, simvastatin sodium salt, pravastatin sodium salt, lovastatin, fluvastatin sodium salt, mevastatin, mevastatin sodium salt, and atorvastatin calcium salt were dissolved in dimethyl sulfoxide 1% (vol/vol) in RPMI. Twofold serial dilutions, with final concentrations ranging from 1.5 μM to 200 μM, were prepared in dimethyl sulfoxide 1% in RPMI and distributed into Falcon 96-well plates just before use. The isotopic microdrug susceptibility test used was described previously (10). Table ​Table11 presents the 50% inhibitory concentrations (IC50) of the different statins for P. falciparum. Simvastatin, fluvastatin, lovastatin, and atorvastatin, in the salt active forms, are more active than simvastatin, mevastatin, and lovastatin, in the lactone form. Pravastatin and mevastatin sodium or potassium salts are inactive against P. falciparum (>200 μM). The results indicate that susceptibility to the salts of simvastatin, fluvastatin, lovastatin, and mevastatin is not dependent on the status of chloroquine resistance. The results observed with the simvastatin salt were similar to those reported by other authors (5). Atorvastatin salt, in the range of 5 to 12 μM, is 10-fold more active against P. falciparum than the other salts. Atorvastatin IC90s ranged from 14.8 to 39 μM. The activity of atorvastatin is independent of the status of chloroquine resistance (4.8 to 5.8 μM against chloroquine-resistant strains versus 5.3 to 11.8 μM for the susceptible strains). TABLE 1. In vitro activities of statins against chloroquine-susceptible (3D7, D6, and IMT031) and chloroquine-resistant (W2, Bre1, and FCR3) P. falciparum strains The chemical structures of simvastatin, lovastatin, mevastatin, and pravastatin are closely related. Those of fluvastatin and atorvastatin are very different from the others. The structural differences between atorvastatin and the other statins could explain differential activity. However, we cannot rule out the action of calcium in the differential activity of atorvastatin. Multiple daily doses of 2.5 to 80 mg of atorvastatin produced steady-state maximum plasma concentrations of 1.95 to 252 μg liter−1 (in the range of 0.2 to 0.3 μM for the maximum) (1). In L6 cells (rat skeletal muscle cell line), atorvastatin at 100 μM induced death in 27% of the cells (7). Although the atorvastatin IC50 for P. falciparum exceeds these reported plasma concentrations, it may be below toxic concentrations. Parasites treated with mevastatin show depressed biosynthesis of dolichol and isoprenoid pyrophosphate (4). In addition, mevastatin decreases the viability of cells by inhibiting proteasome activity. Atorvastatin is an inhibitor for phosphoglycoprotein, an efflux protein, and may be a substrate for this transporter as well (6). A phosphoglycoprotein in P. falciparum, Pgh1, is implicated in quinoline resistance. In conclusion, the present observation suggests that atorvastatin is a good candidate for further studies on the use of statins in malaria treatment.

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

BackgroundAs 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.MethodsThe 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).ResultsAfter 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).ConclusionsThe 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.

Absence of Association between Piperaquine In Vitro Responses and Polymorphisms in the pfcrt, pfmdr1, pfmrp, and pfnhe Genes in Plasmodium falciparum

ABSTRACT 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 (r2 = 0.17; P = 0.0495) and between the responses to PPQ and DOX (r2 = 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 pfnhe-1 genes. There was an absence of cross-resistance with quinolines, and the IC50s 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.

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

BackgroundAs 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.MethodsThe 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.ResultsThe 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%.ConclusionsSince 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.

In vitro susceptibility to quinine and microsatellite variations of the Plasmodium falciparum Na+/H+ exchanger (Pfnhe-1) gene: the absence of association in clinical isolates from the Republic of Congo

BackgroundQuinine is still recommended as an effective therapy for severe cases of Plasmodium falciparum malaria, but the parasite has developed resistance to the drug in some cases. Investigations into the genetic basis for quinine resistance (QNR) suggest that QNR is complex and involves several genes, with either an additive or a pairwise effect. The results obtained when assessing one of these genes, the plasmodial Na+/H+ exchanger, Pfnhe-1, were found to depend upon the geographic origin of the parasite strain. Most of the associations identified have been made in Asian strains; in contrast, in African strains, the influence of Pfnhe on QNR is not apparent. However, a recent study carried out in Kenya did show a significant association between a Pfnhe polymorphism and QNR. As genetic differences may exist across the African continent, more field data are needed to determine if this association exists in other African regions. In the present study, association between Pfnhe and QNR is investigated in a series of isolates from central Africa.MethodsThe sequence analysis of the polymorphisms at the Pfnhe-1 ms4760 microsatellite and the evaluation of in vitro quinine susceptibility (by isotopic assay) were conducted in 74 P. falciparum isolates from the Republic of Congo.ResultsPolymorphisms in the number of DNNND or NHNDNHNNDDD repeats in the Pfnhe-1 ms4760 microsatellite were not associated with quinine susceptibility.ConclusionsThe polymorphism in the microsatellite ms4760 in Pfnhe-1 that cannot be used to monitor quinine response in the regions of the Republic of Congo, where the isolates came from. This finding suggests that there exists a genetic background associated with geographic area for the association that will prevent the use of Pfnhe as a molecular marker for QNR. The contribution of Pfnhe to the in vitro response to quinine remains to be assessed in other regions, including in countries with different levels of drug pressure.

In Vitro Activity of Proveblue (Methylene Blue) on Plasmodium falciparum Strains Resistant to Standard Antimalarial Drugs

ABSTRACT The geometric mean 50% inhibitory concentration (IC50) for Proveblue, a methylene blue complying with the European Pharmacopoeia, was more active on 23 P. falciparum strains than chloroquine, quinine, mefloquine, monodesethylamodiaquine, and lumefantrine. We did not find significant associations between the Proveblue IC50 and polymorphisms in the pfcrt, pfmdr1, pfmdr2, pfmrp, and pfnhe-1 genes or the copy numbers of the pfmdr1 and pfmdr2 genes, all of which are involved in antimalarial resistance.

Atorvastatin Is a Promising Partner for Antimalarial Drugs in Treatment of Plasmodium falciparum Malaria

ABSTRACT 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 (IC50s) 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 IC50s 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) and is not subject to the pH modification induced by the P. falciparum NHE-1 protein. The absence of in vitro cross-resistance between AVA and chloroquine, quinine, mefloquine, monodesethylamodiaquine, lumefantrine, dihydroartemisinin, atovaquone, and doxycycline argues that these antimalarial drugs could potentially be paired with AVA as a treatment for malaria. In conclusion, the present observations suggest that AVA is a good candidate for further studies on the use of statins in association with drugs known to have activities against the malaria parasite.