Correspondence
Low-dose primaquine for falciparum malaria We read with interest Alice Eziefula and colleagues’ report of the results of primaquine gametocidal treatment for falciparum malaria in Ugandan children.1 However, the efficacy of primaquine is totally dependent on its being metabolised by CYP2D6 to its active metabolites. Recently, issues of ethnic differences in the prevalence of non-functioning alleles of CYP2D6 (termed the low metaboliser phenotype) have been called into greater focus. Bennet and coworkers2 have shown in a phase 1 study that low metaboliser and even intermediate metaboliser phenotypes were associated with failure of primaquine treatment. Some people have supermetaboliser phenotypes, in which the efficacy of low doses of primaquine may be enhanced. The prevalence of both low metaboliser and supermetaboliser phenotypes is undetermined in many populations. For example, 12·5% of South African Xhosa people seem to belong to one of these extreme CYP2D6 response groups. 3 Other studies 4 have shown that these phenotypes vary in frequency by an order of magnitude between Africa, Asia, and Oceania. Eziefula and colleagues have shown that even with high doses of primaquine there was still a 5·7% failure rate. The level of CYP2D6 inactive mutations in the Ugandan population is unknown; however, this failure group might represent the low primaquine metabolisers. Altogether, the lesson that can be learnt from these studies is that an extrapolation from primaquine dosefinding studies, done in ethnically unique, homogenous populations (such as in the study of Eziefula and colleagues), cannot be applied to other populations without data about their CYP2D6 genotype. Furthermore, the importance of primaquine therapy lies in its function as the most potent gametocidal drug, and therefore is a www.thelancet.com/infection Vol 14 June 2014
major part of the malaria eradication campaign. The fact that a substantial percentage of some populations might not respond to primaquine, regardless of its dose, may hamper efforts to eradicate malaria. A new gametocidal 8-aminoquinoline drug that circumvents CYP2D6 dependent metabolism is greatly needed. We declare no competing interests.
Eyal Meltzer, *Eli Schwartz [email protected] The Center for Geographic Medicine and Department of Medicine C, The Chaim Sheba Medical Center, Tel Hashomer, 52621 Israel 1
2
3
4
Eziefula AC, Bousema T, Yeung S, et al. Single dose primaquine for clearance of Plasmodium falciparum gametocytes in children with uncomplicated malaria in Uganda: a randomised, controlled, double-blind, dose-ranging trial. Lancet Infect Dis 2014; 14: 130–39. Bennett JW, Pybus BS, Yadava A, et al. Primaquine failure and cytochrome P-450 2D6 in Plasmodium vivax malaria. N Engl J Med 2013; 369: 1381–82. Wright GE, Niehaus DJ, Drogemoller BI, Koen L, Gaedigk A, Warnich L. Elucidation of CYP2D6 genetic diversity in a unique African population: implications for the future application of pharmacogenetics in the Xhosa population. Ann Hum Genet 2010; 74: 340–50. Sistonen J, Sajantila A, Lao O, Corander J, Barbujani G, Fuselli S. CYP2D6 worldwide genetic variation shows high frequency of altered activity variants and no continental structure. Pharmacogenet Genomics 2007; 17: 93–101.
A study by Alice Eziefula and colleagues1 has shown that low-dose primaquine (0·4 mg/kg) is equally effective against gametocytes of Plasmodium falciparum as the previously recommended dose (0·75 mg/kg). A Comment by Richard Steketee and Feiko ter Kuile2 discussed the practical issues related to the use of low doses of primaquine and its timing during the course of treatment. All these issues are important for future studies so that more evidence can be generated regarding its safety and efficacy. Recent WHO guidelines3 also recommend use of low dose of primaquine (0·25 mg/kg) for treatment of P falciparum malaria. A low dose is recommended to provide better coverage of the target population. It has been documented4 that low-dose
primaquine is safely tolerated by G6PD-deficient individuals. However, suboptimum doses of antimalarial drugs can lead to drug resistance. Thus, an increase in the use of primaquine at low doses may lead to an increase in drug resistance. Therefore, user-friendly drug sensitivity assays5 are required to monitor the development of resistance to primaquine and other antimalarial drugs. Low-dose primaquine might be safe for patients but it could also provide safe shelter for the parasite. We declare no competing interests.
Kapil Goyal, Hargobinder Kaur, *Rakesh Sehgal [email protected] Department of Medical Parasitology, Postgraduate Institute of Medical Education and Research, Chandigarh-160012, India 1
2
3
4
5
Eziefula AC, Bousema T, Yeung S, et al. Single dose primaquine for clearance of Plasmodium falciparum gametocytes in children with uncomplicated malaria in Uganda: a randomised, controlled, double-blind, dose-ranging trial. Lancet Infect Dis 2014; 14: 130–39. Steketee RW, ter Kuile F. Single low-dose primaquine to reduce malaria transmission. Lancet Infect Dis 2014; 14: 91–92. WHO. Updated WHO policy recommendation (October 2012): single dose primaquine as a gametocytocide in Plasmodium falciparum malaria. October, 2012. Geneva: World Health Organization, 2012. WHO. WHO Evidence Review Group: the safety and effectiveness of single dose primaquine as a P. falciparum gametocytocide. http://www. who.int/malaria/mpac/sep2012/primaquine_ single_dose_pf_erg_meeting_report_ aug2012.pdf (accessed Sept 19, 2013). D’Alessandro S, Silvestrini F, Dechering K, et al. A Plasmodium falciparum screening assay for anti-gametocyte drugs based on parasite lactate dehydrogenase detection. J Antimicrob Chemother 2013; 68: 2048–58.
Artemisinin resistance in Plasmodium falciparum We reported in The Lancet Infectious Diseases1 that survival of Plasmodium falciparum differs significantly between fast-clearing and slow-clearing parasites isolated from patients with malaria treated with an artemisinin in western Cambodia (10·88% vs 0·23%, p=0·007). Of the 26 parasites tested, however, we found that four gave discordant results. Of the 13 fast-clearing parasites, three had high survival rates 449
Correspondence
Proportion of viable parasites (%)
100
10
1
0·1 Wild-type Arg539Thr Cys580Tyr Tyr493His
0·01
0·001 Fast
Slow Parasite clearance
and slow-clearing parasites with concordant survival rates had wildtype or mutant alleles (Tyr493His and Cys580Tyr), respectively. These data suggest that parasite survival rates in the ring-stage survival assay are more relevant than parasite clearance half-lives for identifying artemisinin-resistant P falciparum, and further validate K13-propeller polymorphisms as a molecular marker of artemisinin resistance in vitro and in vivo. We declare no competing interests.
Figure: Association between parasite survival according to the in-vitro ring-stage survival assay (0–3 h) and parasite clearance in patients treated with artemisinin-based combination therapy, by K13-propeller genotype
(5·30%, 19·32%, and 51·39%) but a resistant stage-dependent pattern (decrease in survival as parasites matured of 1·2%, 17·3%, and 50·2%, respectively). We postulated that these three parasites were indeed artemisinin-resistant, but that their clearance was fast because circulating parasites seen in blood films were predominantly older, drug-sensitive rings at the time the patients received artemisinin drugs. Of the 13 slow-clearing parasites, one had a paradoxically low survival (0·16%). We believe that this parasite was in fact artemisinin-sensitive, but that its clearance was slow because the patient had low levels of parasite-clearing immunity2 or a poor pharmacokinetics profile. We reported K13-propeller polymorphism as a new molecular marker for artemisinin-resistant P falciparum malaria,3 and postulated that K13propeller genotypes would definitively resolve the four discordant results. To test this possibility we genotyped all 26 parasites and found that the three parasites with discordantly high survival rates each carry a different mutant K13-propeller allele (Tyr493His, Cys580Tyr, and Arg539Thr), and that the parasite with a discordantly low survival rate carried a wildtype allele (figure). As expected, we also found that all the fast-clearing 450
Chanaki Amaratunga, Benoit Witkowski, Nimol Khim, Didier Menard, *Rick M Fairhurst [email protected] Laboratory of Malaria and Vector Research, NIAID/National Institutes of Health, Rockville, MD 20852, USA (CA, RMF); and Malaria Molecular Epidemiology Unit, Institut Pasteur du Cambodge, Phnom Penh, Cambodia (BW, NK, DM) 1
2
3
Witkowski B, Amaratunga C, Khim N, et al. Novel phenotypic assays for the detection of artemisinin-resistant Plasmodium falciparum malaria in Cambodia: in-vitro and ex-vivo drug-response studies. Lancet Infect Dis 2013; 13: 1043–49. Lopera-Mesa TM, Doumbia S, Chiang S, et al. Plasmodium falciparum clearance rates in response to artesunate in Malian children with malaria: effect of acquired immunity. J Infect Dis 2013; 207: 1655–63. Ariey F, Witkowski B, Amaratunga C, et al. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria. Nature 2014; 505: 50–55.
monotherapy with artemisinin drugs for treatment of falciparum malaria— particularly the chloroquine-resistant strain—is 5–7 days.2 A study of patients treated with dihydroartemisinin (60 mg) daily (120 mg on day 1) for 3, 5, or 7 days showed no recrudescences in the 7 day group, compared with recrudescence in 66% of patients in the 5 day group and 52% in the 3 day group. Therefore, it was recommended that the 7 day course, with a total dose of 480 mg of dihydroartemisin, is the optimum regimen for treatment of P falciparum infection.3 Another feature of clinical malaria management in China is the quarantine of patients during treatment to prevent transmission and hamper the spread of resistance. Combination of artemisinin drugs with other antimalarial treatments gives more effective treatment at the early stage of malaria infections, although, it is becoming clear that the number of treatment days has a major impact on efficacy of artemisinin treatment either as monotherapy or in combination, especially if ineffective partner drugs are used.4 Although we do not have evidence to demonstrate this hypothesis so far, increasing the duration of artemisinin treatment and use of efficacious partner drugs could sustain the efficacy of artemisinin combination therapies. We declare no competing interests.
Benoit Witkowski and colleagues1 reported a simple and rapid in vitro method to detect artemisinin resistance of P falciparum. They show that the slow clearance of parasites by artemisinin combination therapies is a result of a drop in susceptibility among young ring form parasites whereas susceptibility is maintained in mature stages. However, the clinical relevance of this finding to treatment failure is unclear since these infections could still be successfully treated with artemisinin combination therapies. Experiences in China also suggest that such resistance could be overcome if the right strategies are used. In China, the recommended duration of
*Zhao-Rong Lun, Pedro E Ferreira, Lin-Chun Fu [email protected] Key Laboratory of Gene Engineering of Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Key Laboratory of Tropical Disease Control, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou 510275, China (ZRL); School of Biological Sciences, Nanyang Technological University, Singapore (PEF); and Tropical Medicine Institute, Guangzhou University of Chinese Medicine, Guangzhou, China (LCF) 1
2
Witkowski B, Amaratunga C, Khim N, et al. Novel phenotypic assays for the detection of artemisinin-resistant Plasmodium falciparum malaria in Cambodia: in-vitro and ex-vivo drug-response studies. Lancet Infect Dis 2013; 13: 1043–49. The Ministry of Health of China. National guideline for use and regimen of antimalarials. Chin J Parasit Dis Control 2002; 15: 129–30.
www.thelancet.com/infection Vol 14 June 2014
Low-dose primaquine for falciparum malaria We read with interest Alice Eziefula and colleagues’ report of the results of primaquine gametocidal treatment for falciparum malaria in Ugandan children.1 However, the efficacy of primaquine is totally dependent on its being metabolised by CYP2D6 to its active metabolites. Recently, issues of ethnic differences in the prevalence of non-functioning alleles of CYP2D6 (termed the low metaboliser phenotype) have been called into greater focus. Bennet and coworkers2 have shown in a phase 1 study that low metaboliser and even intermediate metaboliser phenotypes were associated with failure of primaquine treatment. Some people have supermetaboliser phenotypes, in which the efficacy of low doses of primaquine may be enhanced. The prevalence of both low metaboliser and supermetaboliser phenotypes is undetermined in many populations. For example, 12·5% of South African Xhosa people seem to belong to one of these extreme CYP2D6 response groups. 3 Other studies 4 have shown that these phenotypes vary in frequency by an order of magnitude between Africa, Asia, and Oceania. Eziefula and colleagues have shown that even with high doses of primaquine there was still a 5·7% failure rate. The level of CYP2D6 inactive mutations in the Ugandan population is unknown; however, this failure group might represent the low primaquine metabolisers. Altogether, the lesson that can be learnt from these studies is that an extrapolation from primaquine dosefinding studies, done in ethnically unique, homogenous populations (such as in the study of Eziefula and colleagues), cannot be applied to other populations without data about their CYP2D6 genotype. Furthermore, the importance of primaquine therapy lies in its function as the most potent gametocidal drug, and therefore is a www.thelancet.com/infection Vol 14 June 2014
major part of the malaria eradication campaign. The fact that a substantial percentage of some populations might not respond to primaquine, regardless of its dose, may hamper efforts to eradicate malaria. A new gametocidal 8-aminoquinoline drug that circumvents CYP2D6 dependent metabolism is greatly needed. We declare no competing interests.
Eyal Meltzer, *Eli Schwartz [email protected] The Center for Geographic Medicine and Department of Medicine C, The Chaim Sheba Medical Center, Tel Hashomer, 52621 Israel 1
2
3
4
Eziefula AC, Bousema T, Yeung S, et al. Single dose primaquine for clearance of Plasmodium falciparum gametocytes in children with uncomplicated malaria in Uganda: a randomised, controlled, double-blind, dose-ranging trial. Lancet Infect Dis 2014; 14: 130–39. Bennett JW, Pybus BS, Yadava A, et al. Primaquine failure and cytochrome P-450 2D6 in Plasmodium vivax malaria. N Engl J Med 2013; 369: 1381–82. Wright GE, Niehaus DJ, Drogemoller BI, Koen L, Gaedigk A, Warnich L. Elucidation of CYP2D6 genetic diversity in a unique African population: implications for the future application of pharmacogenetics in the Xhosa population. Ann Hum Genet 2010; 74: 340–50. Sistonen J, Sajantila A, Lao O, Corander J, Barbujani G, Fuselli S. CYP2D6 worldwide genetic variation shows high frequency of altered activity variants and no continental structure. Pharmacogenet Genomics 2007; 17: 93–101.
A study by Alice Eziefula and colleagues1 has shown that low-dose primaquine (0·4 mg/kg) is equally effective against gametocytes of Plasmodium falciparum as the previously recommended dose (0·75 mg/kg). A Comment by Richard Steketee and Feiko ter Kuile2 discussed the practical issues related to the use of low doses of primaquine and its timing during the course of treatment. All these issues are important for future studies so that more evidence can be generated regarding its safety and efficacy. Recent WHO guidelines3 also recommend use of low dose of primaquine (0·25 mg/kg) for treatment of P falciparum malaria. A low dose is recommended to provide better coverage of the target population. It has been documented4 that low-dose
primaquine is safely tolerated by G6PD-deficient individuals. However, suboptimum doses of antimalarial drugs can lead to drug resistance. Thus, an increase in the use of primaquine at low doses may lead to an increase in drug resistance. Therefore, user-friendly drug sensitivity assays5 are required to monitor the development of resistance to primaquine and other antimalarial drugs. Low-dose primaquine might be safe for patients but it could also provide safe shelter for the parasite. We declare no competing interests.
Kapil Goyal, Hargobinder Kaur, *Rakesh Sehgal [email protected] Department of Medical Parasitology, Postgraduate Institute of Medical Education and Research, Chandigarh-160012, India 1
2
3
4
5
Eziefula AC, Bousema T, Yeung S, et al. Single dose primaquine for clearance of Plasmodium falciparum gametocytes in children with uncomplicated malaria in Uganda: a randomised, controlled, double-blind, dose-ranging trial. Lancet Infect Dis 2014; 14: 130–39. Steketee RW, ter Kuile F. Single low-dose primaquine to reduce malaria transmission. Lancet Infect Dis 2014; 14: 91–92. WHO. Updated WHO policy recommendation (October 2012): single dose primaquine as a gametocytocide in Plasmodium falciparum malaria. October, 2012. Geneva: World Health Organization, 2012. WHO. WHO Evidence Review Group: the safety and effectiveness of single dose primaquine as a P. falciparum gametocytocide. http://www. who.int/malaria/mpac/sep2012/primaquine_ single_dose_pf_erg_meeting_report_ aug2012.pdf (accessed Sept 19, 2013). D’Alessandro S, Silvestrini F, Dechering K, et al. A Plasmodium falciparum screening assay for anti-gametocyte drugs based on parasite lactate dehydrogenase detection. J Antimicrob Chemother 2013; 68: 2048–58.
Artemisinin resistance in Plasmodium falciparum We reported in The Lancet Infectious Diseases1 that survival of Plasmodium falciparum differs significantly between fast-clearing and slow-clearing parasites isolated from patients with malaria treated with an artemisinin in western Cambodia (10·88% vs 0·23%, p=0·007). Of the 26 parasites tested, however, we found that four gave discordant results. Of the 13 fast-clearing parasites, three had high survival rates 449
Correspondence
Proportion of viable parasites (%)
100
10
1
0·1 Wild-type Arg539Thr Cys580Tyr Tyr493His
0·01
0·001 Fast
Slow Parasite clearance
and slow-clearing parasites with concordant survival rates had wildtype or mutant alleles (Tyr493His and Cys580Tyr), respectively. These data suggest that parasite survival rates in the ring-stage survival assay are more relevant than parasite clearance half-lives for identifying artemisinin-resistant P falciparum, and further validate K13-propeller polymorphisms as a molecular marker of artemisinin resistance in vitro and in vivo. We declare no competing interests.
Figure: Association between parasite survival according to the in-vitro ring-stage survival assay (0–3 h) and parasite clearance in patients treated with artemisinin-based combination therapy, by K13-propeller genotype
(5·30%, 19·32%, and 51·39%) but a resistant stage-dependent pattern (decrease in survival as parasites matured of 1·2%, 17·3%, and 50·2%, respectively). We postulated that these three parasites were indeed artemisinin-resistant, but that their clearance was fast because circulating parasites seen in blood films were predominantly older, drug-sensitive rings at the time the patients received artemisinin drugs. Of the 13 slow-clearing parasites, one had a paradoxically low survival (0·16%). We believe that this parasite was in fact artemisinin-sensitive, but that its clearance was slow because the patient had low levels of parasite-clearing immunity2 or a poor pharmacokinetics profile. We reported K13-propeller polymorphism as a new molecular marker for artemisinin-resistant P falciparum malaria,3 and postulated that K13propeller genotypes would definitively resolve the four discordant results. To test this possibility we genotyped all 26 parasites and found that the three parasites with discordantly high survival rates each carry a different mutant K13-propeller allele (Tyr493His, Cys580Tyr, and Arg539Thr), and that the parasite with a discordantly low survival rate carried a wildtype allele (figure). As expected, we also found that all the fast-clearing 450
Chanaki Amaratunga, Benoit Witkowski, Nimol Khim, Didier Menard, *Rick M Fairhurst [email protected] Laboratory of Malaria and Vector Research, NIAID/National Institutes of Health, Rockville, MD 20852, USA (CA, RMF); and Malaria Molecular Epidemiology Unit, Institut Pasteur du Cambodge, Phnom Penh, Cambodia (BW, NK, DM) 1
2
3
Witkowski B, Amaratunga C, Khim N, et al. Novel phenotypic assays for the detection of artemisinin-resistant Plasmodium falciparum malaria in Cambodia: in-vitro and ex-vivo drug-response studies. Lancet Infect Dis 2013; 13: 1043–49. Lopera-Mesa TM, Doumbia S, Chiang S, et al. Plasmodium falciparum clearance rates in response to artesunate in Malian children with malaria: effect of acquired immunity. J Infect Dis 2013; 207: 1655–63. Ariey F, Witkowski B, Amaratunga C, et al. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria. Nature 2014; 505: 50–55.
monotherapy with artemisinin drugs for treatment of falciparum malaria— particularly the chloroquine-resistant strain—is 5–7 days.2 A study of patients treated with dihydroartemisinin (60 mg) daily (120 mg on day 1) for 3, 5, or 7 days showed no recrudescences in the 7 day group, compared with recrudescence in 66% of patients in the 5 day group and 52% in the 3 day group. Therefore, it was recommended that the 7 day course, with a total dose of 480 mg of dihydroartemisin, is the optimum regimen for treatment of P falciparum infection.3 Another feature of clinical malaria management in China is the quarantine of patients during treatment to prevent transmission and hamper the spread of resistance. Combination of artemisinin drugs with other antimalarial treatments gives more effective treatment at the early stage of malaria infections, although, it is becoming clear that the number of treatment days has a major impact on efficacy of artemisinin treatment either as monotherapy or in combination, especially if ineffective partner drugs are used.4 Although we do not have evidence to demonstrate this hypothesis so far, increasing the duration of artemisinin treatment and use of efficacious partner drugs could sustain the efficacy of artemisinin combination therapies. We declare no competing interests.
Benoit Witkowski and colleagues1 reported a simple and rapid in vitro method to detect artemisinin resistance of P falciparum. They show that the slow clearance of parasites by artemisinin combination therapies is a result of a drop in susceptibility among young ring form parasites whereas susceptibility is maintained in mature stages. However, the clinical relevance of this finding to treatment failure is unclear since these infections could still be successfully treated with artemisinin combination therapies. Experiences in China also suggest that such resistance could be overcome if the right strategies are used. In China, the recommended duration of
*Zhao-Rong Lun, Pedro E Ferreira, Lin-Chun Fu [email protected] Key Laboratory of Gene Engineering of Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Key Laboratory of Tropical Disease Control, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou 510275, China (ZRL); School of Biological Sciences, Nanyang Technological University, Singapore (PEF); and Tropical Medicine Institute, Guangzhou University of Chinese Medicine, Guangzhou, China (LCF) 1
2
Witkowski B, Amaratunga C, Khim N, et al. Novel phenotypic assays for the detection of artemisinin-resistant Plasmodium falciparum malaria in Cambodia: in-vitro and ex-vivo drug-response studies. Lancet Infect Dis 2013; 13: 1043–49. The Ministry of Health of China. National guideline for use and regimen of antimalarials. Chin J Parasit Dis Control 2002; 15: 129–30.
www.thelancet.com/infection Vol 14 June 2014
