Resistance to Dihydroartemisinin
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《传染病的形成》
Hpital Bichat Claude Bernard, Assistance Publique–Hpitaux de Paris, Paris, France
Universite Paris 5, Paris, France
Hpital Avicenne, Bobigny, France
Universite Paris 13, Bobigny, France
We assessed the in vitro susceptibility to dihydroartemisinin (dhART), the biologically active metabolite of artemisinin derivatives, of P. falciparum isolates from travelers returning to France from various African countries during 2004–2006. In addition, we searched for polymorphism in the P. falciparum adenosine triphosphatase-6 (PfATPase6) gene, which was reported to be associated with in vitro artemether resistance (5). We also studied polymorphism (a 3-bp indel) in the gene of the ABC transporter G7, which was reported in 2005 to be associated with in vitro response to artesunate (6).
Determination of in vitro dhART susceptibility by using the isotopic semimicrotest method (7) was successful for 397 isolates. The most represented countries were Cameroon (17%), Cotê d'Ivoire(14.5%), Mali (12%), Comoros Islands (8.5%), and Senegal (6.5%). Patients were <75 years of age (mean 31, SD 17 years), and the male:female ratio was 1.5:1. The 50% inhibitory concentration (IC50) values ranged from 0.02 to 31.8 nmol/L, with a geometric mean of 1.31 nmol/L and a median of 0.68 nmol/L. IC50 values were <1 nmol/L for 264 isolates, 1–10 nmol/L for 127, and >10 nmol/L for 6. Thus, some isolates showed a diminished susceptibility to dhART, but only 1 isolate had an IC50 >30 nmol/L (31.8 nmol/L).
DNA sequencing of 900-bp and 240-bp PCR products, including the 769 and the 243/263 PfATPase6 codons, respectively, was performed in a subsample of 154 isolates. All isolates had the S769 wild codon except 1 susceptible isolate (IC50 = 0.83 nmol/L), which had a S769N mutant type codon (Table). We found no polymorphism in codon 263. This position may be scrutinized to monitor anticipated artemisinin resistance, according to a recently published structure-function study (8). Conversely, we found 2 isolates that had IC50 values of 4.2 nmol/L and 6.4 nmol/L and that showed an H243Y mutant type codon. The role of such a polymorphism appears unclear. We found no association between the 3-bp indel in G7 and in vitro dhART susceptibility because mutants were regularly distributed in highly susceptible isolates and in isolates having a diminished susceptibility.
For our samples obtained during 2004–2006, the geometric mean IC50 value for dhART was very close to values found in Cameroon during 1997–1998 (mean dhART IC50 = 1.11 nmol/L) (9), in Senegal in 2001 (mean artemether IC50 = 1.3 nmol/L) (5), and in Republic of Congo during 2005–2006 (mean dhART IC50 = 1.02 nmol/L) (10). Ringwald et al. observed a narrower range of IC50s, but their series included only 65 samples (9). Previous comparisons between ACs suggested that dhART is 1.7 times more potent than artemether against P. falciparum (9). Thus, the highest IC50 value for artemether observed by Jambou et al. in Senegal (44.7 nmol/L) (5) is comparable to the highest IC50 value for dhART in our series (31.8 nmol/L). The resistance levels of ACs are still undefined. For artemether, Jambou et al. used a threshold of 30 nmol/L to evaluate the association between the S769N mutation and in vitro susceptibility. The presence of ATPase6 S769N was not associated with diminished in vitro susceptibility in our series. Conversely, the only S769N mutant that we observed was found in a fully susceptible isolate. Thus, we confirmed that polymorphism exists in this gene in positions 769 and 243, but we did not prove an association between these point mutations and resistance to ACs. Similarly, our results did not support the hypothesis of an association between the 3-bp indel in G7 and resistance to ACs.
ACs, considered the most important class of antimalarial drugs, merit close surveillance for susceptibility. Continued monitoring of the efficacy of their associated partner drugs also appears to be essential.
Acknowledgments
We thank the regular correspondents of the Centre National de Reference du Paludisme. Financial support was provided by the French Ministry of Health (Direction Generale de la Sante). SC received a thesis fellowship from the French Research Ministry.
References
Attaran A, Barnes KI, Curtis C, D'Alessandro U, Fanello C, Galinski MR, et al. WHO, the Global Fund, and medical malpractice in malaria treatment. Lancet. 2004;363:237–40.
Mutabingwa TK. Artemisinin-based combination therapies (ACTs): best hope for malaria treatment but inaccessible to the needy! Acta Trop. 2005;95:305–15.
Ittarat W, Pickard AL, Rattanasinganchan P, Wilairatana P, Looareesuwan S, Emery K, et al. Recrudescence in artesunate-treated patients with falciparum malaria is dependent on parasite burden not on parasite factors. Am J Trop Med Hyg. 2003;68:147–52.
Grandesso F, Hagerman A, Kamara S, Lam E, Checchi F, Balkan S, et al. Low efficacy of the combination artesunate plus amodiaquine for uncomplicated falciparum malaria among children under 5 in Kailahun, Sierra Leone. Trop Med Int Health. 2006;11:1017–21.
Jambou R, Legrand E, Niang M, Khim N, Lim P, Volney B, et al. Resistance of Plasmodium falciparum field isolates to in-vitro artemether and point mutations of the SERCA-type PfATPase6. Lancet. 2005;366:1960–3.
Anderson TJC, Nair S, Qin H, Singlam S, Brockman A, Paiphun L, et al. Are transporter genes other than the chloroquine resistance locus (pfcrt) and multidrug resistance gene (pfmdr) associated with antimalarial drug resistance Antimicrob Agents Chemother. 2005;49:2180–8.
Le Bras J, Andrieu B, Hatin I, Savel J, Coulaud JP. Plasmodium falciparum: interpretation of the semi-microtest of in vitro chemosensitivity by H3-hypoxanthine incorporation [in French]. Pathol Biol. 1984;32:463–6.
Uhlemann AC, Cameron A, Eckstein-Ludwig U, Fischbarg J, Iserovich P, Zuniga FA, et al. A single amino acid residue can determine the sensitivity of SERCAs to artemisinins. Nat Struct Mol Biol. 2005;12:628–9.
Ringwald P, Bickii J, Basco LK. In vitro activity of dihydroartemisinin against clinical isolates of Plasmodium falciparum in Yaounde, Cameroon. Am J Trop Med Hyg. 1999;61:187–92.
Pradines B, Hovette P, Fusai T, Atanda HL, Baret E, Cheval P, et al. Prevalence of in vitro resistance to eleven standard or new antimalarial drugs among Plasmodium falciparum isolates from Pointe-Noire, Republic of the Congo. J Clin Microbiol. 2006;44:2404–8.(Sandrine Cojean, Veroniqu)
Universite Paris 5, Paris, France
Hpital Avicenne, Bobigny, France
Universite Paris 13, Bobigny, France
We assessed the in vitro susceptibility to dihydroartemisinin (dhART), the biologically active metabolite of artemisinin derivatives, of P. falciparum isolates from travelers returning to France from various African countries during 2004–2006. In addition, we searched for polymorphism in the P. falciparum adenosine triphosphatase-6 (PfATPase6) gene, which was reported to be associated with in vitro artemether resistance (5). We also studied polymorphism (a 3-bp indel) in the gene of the ABC transporter G7, which was reported in 2005 to be associated with in vitro response to artesunate (6).
Determination of in vitro dhART susceptibility by using the isotopic semimicrotest method (7) was successful for 397 isolates. The most represented countries were Cameroon (17%), Cotê d'Ivoire(14.5%), Mali (12%), Comoros Islands (8.5%), and Senegal (6.5%). Patients were <75 years of age (mean 31, SD 17 years), and the male:female ratio was 1.5:1. The 50% inhibitory concentration (IC50) values ranged from 0.02 to 31.8 nmol/L, with a geometric mean of 1.31 nmol/L and a median of 0.68 nmol/L. IC50 values were <1 nmol/L for 264 isolates, 1–10 nmol/L for 127, and >10 nmol/L for 6. Thus, some isolates showed a diminished susceptibility to dhART, but only 1 isolate had an IC50 >30 nmol/L (31.8 nmol/L).
DNA sequencing of 900-bp and 240-bp PCR products, including the 769 and the 243/263 PfATPase6 codons, respectively, was performed in a subsample of 154 isolates. All isolates had the S769 wild codon except 1 susceptible isolate (IC50 = 0.83 nmol/L), which had a S769N mutant type codon (Table). We found no polymorphism in codon 263. This position may be scrutinized to monitor anticipated artemisinin resistance, according to a recently published structure-function study (8). Conversely, we found 2 isolates that had IC50 values of 4.2 nmol/L and 6.4 nmol/L and that showed an H243Y mutant type codon. The role of such a polymorphism appears unclear. We found no association between the 3-bp indel in G7 and in vitro dhART susceptibility because mutants were regularly distributed in highly susceptible isolates and in isolates having a diminished susceptibility.
For our samples obtained during 2004–2006, the geometric mean IC50 value for dhART was very close to values found in Cameroon during 1997–1998 (mean dhART IC50 = 1.11 nmol/L) (9), in Senegal in 2001 (mean artemether IC50 = 1.3 nmol/L) (5), and in Republic of Congo during 2005–2006 (mean dhART IC50 = 1.02 nmol/L) (10). Ringwald et al. observed a narrower range of IC50s, but their series included only 65 samples (9). Previous comparisons between ACs suggested that dhART is 1.7 times more potent than artemether against P. falciparum (9). Thus, the highest IC50 value for artemether observed by Jambou et al. in Senegal (44.7 nmol/L) (5) is comparable to the highest IC50 value for dhART in our series (31.8 nmol/L). The resistance levels of ACs are still undefined. For artemether, Jambou et al. used a threshold of 30 nmol/L to evaluate the association between the S769N mutation and in vitro susceptibility. The presence of ATPase6 S769N was not associated with diminished in vitro susceptibility in our series. Conversely, the only S769N mutant that we observed was found in a fully susceptible isolate. Thus, we confirmed that polymorphism exists in this gene in positions 769 and 243, but we did not prove an association between these point mutations and resistance to ACs. Similarly, our results did not support the hypothesis of an association between the 3-bp indel in G7 and resistance to ACs.
ACs, considered the most important class of antimalarial drugs, merit close surveillance for susceptibility. Continued monitoring of the efficacy of their associated partner drugs also appears to be essential.
Acknowledgments
We thank the regular correspondents of the Centre National de Reference du Paludisme. Financial support was provided by the French Ministry of Health (Direction Generale de la Sante). SC received a thesis fellowship from the French Research Ministry.
References
Attaran A, Barnes KI, Curtis C, D'Alessandro U, Fanello C, Galinski MR, et al. WHO, the Global Fund, and medical malpractice in malaria treatment. Lancet. 2004;363:237–40.
Mutabingwa TK. Artemisinin-based combination therapies (ACTs): best hope for malaria treatment but inaccessible to the needy! Acta Trop. 2005;95:305–15.
Ittarat W, Pickard AL, Rattanasinganchan P, Wilairatana P, Looareesuwan S, Emery K, et al. Recrudescence in artesunate-treated patients with falciparum malaria is dependent on parasite burden not on parasite factors. Am J Trop Med Hyg. 2003;68:147–52.
Grandesso F, Hagerman A, Kamara S, Lam E, Checchi F, Balkan S, et al. Low efficacy of the combination artesunate plus amodiaquine for uncomplicated falciparum malaria among children under 5 in Kailahun, Sierra Leone. Trop Med Int Health. 2006;11:1017–21.
Jambou R, Legrand E, Niang M, Khim N, Lim P, Volney B, et al. Resistance of Plasmodium falciparum field isolates to in-vitro artemether and point mutations of the SERCA-type PfATPase6. Lancet. 2005;366:1960–3.
Anderson TJC, Nair S, Qin H, Singlam S, Brockman A, Paiphun L, et al. Are transporter genes other than the chloroquine resistance locus (pfcrt) and multidrug resistance gene (pfmdr) associated with antimalarial drug resistance Antimicrob Agents Chemother. 2005;49:2180–8.
Le Bras J, Andrieu B, Hatin I, Savel J, Coulaud JP. Plasmodium falciparum: interpretation of the semi-microtest of in vitro chemosensitivity by H3-hypoxanthine incorporation [in French]. Pathol Biol. 1984;32:463–6.
Uhlemann AC, Cameron A, Eckstein-Ludwig U, Fischbarg J, Iserovich P, Zuniga FA, et al. A single amino acid residue can determine the sensitivity of SERCAs to artemisinins. Nat Struct Mol Biol. 2005;12:628–9.
Ringwald P, Bickii J, Basco LK. In vitro activity of dihydroartemisinin against clinical isolates of Plasmodium falciparum in Yaounde, Cameroon. Am J Trop Med Hyg. 1999;61:187–92.
Pradines B, Hovette P, Fusai T, Atanda HL, Baret E, Cheval P, et al. Prevalence of in vitro resistance to eleven standard or new antimalarial drugs among Plasmodium falciparum isolates from Pointe-Noire, Republic of the Congo. J Clin Microbiol. 2006;44:2404–8.(Sandrine Cojean, Veroniqu)