Artemisinin
Artemisinin and its derivatives have been effectively used to treat Plasmodium falciparum malaria patients. Artemisinins are, among the most potent antimalarial agents, effective against all asexual parasite stages. Despite being the most effective drugs against several stages of malaria parasites, artemisinins are rapidly transformed into inactive metabolite inside the human body (approximately in 1 hour). Combinations of artemisinin with a long-lasting drug, called artemisinin-based combination therapies (ACTs), are recommended for falciparum malaria treatment. Delayed parasite clearance following artemisinin treatment is attributed to poor cure rates and high recrudescence rates for short courses of artemisinin treatment.
Artemisinin resistance is different from other antimalarial drugs since the drugs are currently effective against the parasites. However, in 2009, a study reported the Cambodian P. falciparum patients receiving ACTs and took longer time to clear their parasites (Dondrop, et al, 2009, N Engl J Med). Resistance to artemisinin is characterized by slow parasite clearance in vivo without corresponding reductions on conventional in vitro susceptibility testing. An effective assay for evaluating artemisinin resistance is ring-stage survival assay (RSA).
Several studies demonstrated the association between slow clearing parasites in ACTs and mutations in pfkelch13 gene.
- The parasite clearance half-life in patients with wide type K13 parasite (median 3.30h) is shorter than mutant with C580Y (7.19 h), R539T (6.64h) and Y493H (6.28h). (Ariey F, et al., 2014, Nature)
- Parasite clearance half-life (>5 hours) associated with mutations of the P. falciparum K13 gene after position 440. (Ashley EA, et al., 2014, N Engl J Med)
- Kelch13 mutations that have slow parasite Clearance half-life (P441L, G449A, N458Y, Y493H, R539T, I543T, P553L, R561H, P574L, C580Y and A675V). (Ashley EA, et al., 2014, N Engl J Med)
- Kelch13 mutations (M476I, Y493H, R539T, I543T, C580Y) confer artemisinin resistance , defined in RSA 0-3h using zinc-finger nucleases technique to introduce the mutationin clinical isolates and reference lines in vitro. (Straimer J, et al.,2015,Science.)
- Locus utilized: PF3D7_1343700 (kelch13)
- Codon: codons 349-726
- Field utilized: geno_kelch13
| Amino acid change | Coding | Interpretation | Phenotype |
|---|---|---|---|
| None | geno_kelch13 == "WT" | Wild-type | Sensitive |
| Missing | geno_kelch13 == "-" | Missing | Undetermined/Missing |
| P441L ab, F446Ia, G449Aab, N458Yabe, M476Iacd, Y493Habcd, G538Va, R539Tabcd, I543Tabcd, P553Lab, R561Hab, V568Gab, P574Lab, R575Ka, M579Ia, C580Yabcde, D584Va, P667Ta, F673Ia A675Vab, H719Nab | geno_kelch13 %in% c("P441L", “F446I”, "G449A", "N458Y", "M476I", "Y493H", “G538V”, "R539T", "I543T", "P553L", "R561H", "V568G", "P574L", "C580Y", “D584V”, “P667T”, “F673I”, "A675V”, “H719N”) | Associated mutation with slow parasite clearance t1/2 | Resistant |
| A578Sa, E252Qab | geno_kelch13 %in% c("A578S", "E252Q") | Unassociated with slow parasite clearance t1/2 | Sensitive |
| Other amino acid change | Otherwise | Unknown mutation | Undetermined |
a http://apps.who.int/iris/bitstream/10665/255213/1/WHO-HTM-GMP-2017.9-eng.pdf?ua=1
b Ashley EA, Dhorda M, Fairhurst RM, Amaratunga C, Lim P, Suon S, et al. Spread of artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med. 2014;371(5):411-23.
c Straimer J, Gnadig NF, Witkowski B, Amaratunga C, Duru V, Ramadani AP, et al. Drug resistance. K13-propeller mutations confer artemisinin resistance in Plasmodium falciparum clinical isolates. Science. 2015;347(6220):428-31.
d Ariey F, Witkowski B, Amaratunga C, Beghain J, Langlois AC, Khim N, et al. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria. Nature. 2014;505(7481):50-5.
e Boulle M, Witkowski B, Duru V, Sriprawat K, Nair SK, McDew-White M, et al. Artemisinin-Resistant Plasmodium falciparum K13 Mutant Alleles, Thailand-Myanmar Border. Emerg Infect Dis. 2016;22(8):1503-5
| Reference | Method | Location/Sample size/Genetic background | Finding summary |
|---|---|---|---|
| Ariey F, Witkowski B, Amaratunga C, Beghain J, Langlois AC, Khim N, et al. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria. Nature. 2014;505(7481):50-5. | - Artemisinin-resistant parasite lines were selected under a dose-escalating regimen of artemisinin for 5 years. - Ring-stage survival assay (RSA0-3h) was performed. - Whole genome sequencing was performed to the drug-selected parasites using Illumina platform. - Clinical P. falciparum isolates were collected. - Parasite clearance half-life was determined from the patients treated with artemisinin. |
Drug selection - F32-Tanzania line was used as the parental line. Clinical isolates - Cambodia. (Pursat and Ratanakiri provinces) - 150 parasite isolate from patients during 2009-2010. |
- Whole-genome sequence of the selected artemisinin-resistant parasites identified 8 mutations in 7 genes, including D56V mutation in RPB9-coding gene and M476I mutations in kelch 13 gene. - Parasites with kelch 13 M476I mutation showed increased RSA0-3h compared to that of F32 parental line. - The parasite clearance half-life in parasites with wild-type kelch 13 gene (median 3.30h) was shorter than that of the parasites with kelch 13 mutations; C580Y (7.19 h), R539T (6.64 h) and Y493H (6.28 h). |
| Ashley EA, Dhorda M, Fairhurst RM, Amaratunga C, Lim P, Suon S, et al. Spread of artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med. 2014;371(5):411-23. | - Parasite clearance half-life was determined from peripheral-blood sample. - Polymorphisms of propeller domain in kelch 13 gene were determined by Illumina platform following a PCR assay. |
- Clinical isolates were collected form 1,241 patients (adults and children) during 2011-2013 at 15 study sites in 10 countries; Cambodia (4 sites), Thailand (3 sites), and Laos, Vietnam, Myanmar, Bangladesh, India, Nigeria, Kenya, and the Democratic Republic of Congo (1 site each). | - The mutations in the kelch 13 gene after position 440 which associated with >5 Parasite clearance half-life are P441L, G449A, N458Y, Y493H, R539T, I543T, P553L, R561H, P574L, C580Y and A675V. |
| Phyo AP, Ashley EA, Anderson TJC, Bozdech Z, Carrara VI, Sriprawat K, et al. Declining Efficacy of Artemisinin Combination Therapy Against P. Falciparum Malaria on the Thai-Myanmar Border (2003-2013): The Role of Parasite Genetic Factors. Clin Infect Dis. 2016;63(6):784-91. | - Polymorphisms of propeller domain in kelch 13 gene were determined by ABI 3730 capillary sequencer following a PCR assay. - 3 polymorphic loci (MSP1, MSP2 and GLURF) were used to distinguish recrudescence from reinfection. |
- Clinical isolates were collected from 1,005 patients during 2003-2013 in Northwestern border of Thailand. | - Recurrent isolates had higher percentage of kelch 13 mutation (81/112[72.3%]) than that of admission isolates (314/699[44.9%]). Among recurrent isolates, the percentage of kelch 13 mutation was higher in recrudescent isolates (65/76 [85.5%]) than reinfection isolates (12/24 [50%]). |
| Menard D, Khim N, Beghain J, Adegnika AA, Shafiul-Alam M, Amodu O, et al. A Worldwide Map of Plasmodium falciparum K13-Propeller Polymorphisms. N Engl J Med. 2016;374(25):2453-64 | - The A578S mutation was introduced into kelch 13 gene by using zinc-finger nuclease approach. - Ring-stage survival assay was used to assess in vitro artemisinin susceptibility. |
- Dd2 line was used as a parental line. | - The A578G transgenic parasites were susceptible to artemisinin on RSA. |
| Boulle M, Witkowski B, Duru V, Sriprawat K, Nair SK, McDew-White M, et al. Artemisinin-Resistant Plasmodium falciparum K13 Mutant Alleles, Thailand-Myanmar Border. Emerg Infect Dis. 2016;22(8):1503-5 | - Parasite clearance half-life (PCHL) was determined from peripheral-blood. - In vitro, artemisinin-resistant phenotype was determined by ring-stage survival assay (RSA0-3h). - Kelch 13 propeller polymorphisms were determined from sequencing following a PCR amplification assay. |
- Clinical parasite isolates were collected at the Thailand-Myanmar border during 2011-2013 - Total 33 patients were included in this study based on their parasite clearance half-life outcome. - Artemisinin susceptibility of 25 isolates was determined by RSA0-3h. |
- Total 18 out of 23 isolates from the patients with more than 5 hours PCHL carried kelch 13 mutation. - All except 4 isolates from patients who has PCHL more than 5 hours carried k13 mutant allele (18/23). - C580Y and N458Y mutatations consistently associated with parasite clearance half-life more than 5 hours and RSA value greater than 1%. - Disagreement between PCHL and RSA results was observed in E252Q, P441L, G538V and A675V mutations. |
| Reference | Method | Location/Sample size/Genetic background | Finding summary |
|---|---|---|---|
| Straimer J, Gnadig NF, Witkowski B, Amaratunga C, Duru V, Ramadani AP, et al. Drug resistance. K13-propeller mutations confer artemisinin resistance in Plasmodium falciparum clinical isolates. Science. 2015;347(6220):428-31. | - Zinc-finger nucleases (ZNFs) was used for K13 mutation. The mutant K13 alleles was introduced with human drfr selectable marker and then selected with WR99210. - Ring survival assay (RSA0-3h) |
- Cambodian isolates C580Y (FCB, Dd2, CamWT, Cam2, cam3.II), Y493H (Dd2), M476I (Dd2), R539T (Dd2), I543T (Dd2), M476I (F32-TEM) | - This study used the zinc-finger nucleases technique to introduce (476I, 493H, 539T, 543T, 580Y) and remove (539T, 543T, 580Y) mutations at propeller domain of Kelch 13 in Plasmodium falciparum and assess the effects on RSA0-3h. |
- Ring-stage Survival Assay
- Amodiaquine
- Lumefantrine
- Mefloquine
- Piperaquine
- Artesunate-mefloquine (AS-MQ)
- Dihydroartemisinin-piperaquine (DHA-PPQ)
- Dondorp AM, Nosten F, Yi P, Das D, Phyo AP, Tarning J, et al. Artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med. 2009;361(5):455-67.