Category Archives: Mutations

Presence of k13 561H artemisinin resistance mutations in Plasmodium falciparum infections from Rwanda

Aline Uwimana, Noella Umulisa, Meera Venkatesan, Eric S. Halsey, Tharcisse Munyaneza, Rafiki Madjid Habimana, Ryan Sandford, Leah F. Moriarty, Emily Piercefield, Zhiyong Zhou, Samaly Souza, Ira Goldman, Naomi Lucchi, Brian Ezema, Eldin Talundzic, Daniel Ngamije, Jean-Louis N Mangala, William Brieger, Venkatachalam Udhayakumar, and Aimable Mbituyumuremyi presented a poster on “Presence of k13 561H artemisinin resistance mutations in Plasmodium falciparum infections from Rwanda” at the 68th Annual Meeting of the American Society of Tropical Medicine and Hygiene. Their findings follow.

Artemisinin-based combination therapy (ACT) is the recommended first-line antimalarial for uncomplicated Plasmodium falciparum infection in Rwanda. With the emergence of artemisinin and partner drug resistance in the Greater Mekong sub-region, it is important to characterize the presence of polymorphisms in k13, a gene associated with artemisinin resistance, and in pfmdr1, a gene associated with susceptibility to partner drugs including lumefantrine.

To date, there have been sporadic reports of validated k13 markers in Africa. Adequate efficacy (94-97%) for the ACT artemether-lumefantrine (AL) was found in a therapeutic efficacy study (TES) conducted in three Rwandan sites (Masaka, Rukara and Bugarama) in 2018. TES clinical results are presented in poster LB-5134. Dried blood spots collected from the 2018 TES were characterized for artemisinin resistance-associated k13 molecular markers and 8 flanking microsatellites to assess genetic profile and diversity, along with mutations in pfmdr1.

Methods: DNA was isolated from day 0 and day of recurrence dried blood spots from a 2018 TES of AL conducted in 3 sites in Rwanda and analyzed by Sanger sequencing for resistance markers in the k13 and pfmdr1 genes.

Prevalence of k13 candidate and validated artemisinin resistance markers was calculated using day 0 samples. Presence of k13 markers post-treatment was determined using samples collected on the day of recurrence. 8 flanking microsatellite markers downstream and upstream of k13 were evaluated and compared with previously published results from samples from Thailand1.

Results of Prevalence of k13 561 were derived from HDNA from 219 of 228 day 0 samples and all 37 post-treatment samples were successfully isolated from dried blood spots. 26 of 219 day 0 samples showed presence of the 561H mutation in the k13 gene, a World Health Organization validated marker of artemisinin resistance (ref). 3 of the 26 were mixed infections with wild type (561R/H).

*WHO definitions2: Suspected endemic artemisinin resistance is defined as

  • • ? 10% of patients with a half-life of the parasite clearance slope ? 5 hours after treatment with ACT or artesunate monotherapy; or
  • • ? 5% of patients carrying k13 resistance-confirmed mutations; or
  • • ? 10% of patients with persistent parasitaemia by microscopy at 72 hours
    Confirmed endemic artemisinin resistance is defined as
  • • ? 5% of patients carrying k13 resistance-confirmed mutations, all of whom have been found to have either persistent parasitaemia by microscopy on day 3 or a half-life of the parasite clearance slope ? 5 hours after treatment

Additional k13 data found 8 of 37 post-treatment samples with k13 561H in 4 recrudescences (all also with 561H on day 0) and in 4 reinfections. Candidate k13 resistance marker 469F found in 3 day 0 samples (2 in Rukara, 1 in Bugarama) Candidate k13 resistance markers 441L and 449A found in 1 day 0 sample each (Masaka and Rukara, respectively)

In conclusion, k13 561H, a validated marker of artemisinin resistance, was found at a prevalence of 1-20% amongst 3 TES sites in Rwanda. This is the highest proportion of artemisinin resistance-confirmed k13 mutations reported to date in Africa. The overall efficacy of AL was high in all sites (>90%; see poster LB-5134). However, parasitemia on day 3, a proxy for delayed parasite clearance, ranged from 0-15% across sites. Together with the presence of k13 561H, our results indicate confirmed artemisinin resistance in one site, Masaka, and suspected resistance in another, Rukara.

Flanking microsatellites indicate that the k13 561H mutation likely arose locally as opposed to being introduced from Southeast Asia. k13 mutations are present against a high background prevalence of pfmdr1 N86 and D1246, associated with reduced susceptibility to lumefantrine4.

These results indicate that although AL remains an effective treatment of uncomplicated malaria in Rwanda, artemisinin resistance may be emerging. Continued monitoring and confirmation of suspected resistance is critical. Future studies will include an expansion of TES sites and frequent parasite sampling to assess parasite clearance rates, in addition to molecular analysis.

References

  1. Talundzic et al 2015. Selection and Spread of Artemisinin-Resistant Alleles in Thailand Prior to the Global Artemisinin Resistance Containment Campaign PLoS Pathogens 11(4)
  2. WHO 2017. Status report on artemisinin and ACT resistance
  3. Ishengoma et al 2019. Efficacy and safety of artemether-lumefantrine for the treatment of uncomplicated malaria and prevalence of Pfk13 and Pfmdr1 polymorphisms after a decade of using artemisinin-based combination therapy in mainland Tanzania Malaria Journal 18(1):88.
  4. Venkatesan et al 2014 . Polymorphisms in Plasmodium falciparum chloroquine resistance transporter and multidrug resistance 1 genes: parasite risk factors that affect treatment outcomes for P. falciparum malaria after artemether-lumefantrine and artesunate-amodiaquine. AJTMH 91(4)

Contact information: Dr. Aline Uwimana <aline.uwimana@rbc.gov.rw> and Dr. Meera Venkatesan <mvenkatesan@usaid.gov>

Affiliations: Malaria and Other Parasitic Diseases Division, Rwanda Biomedical Centre, Kigali, Rwanda; Maternal and Child Survival Program/JHPIEGO, Baltimore MD, USA; US President’s Malaria Initiative, Washington DC, USA; US President’s Malaria Initiative, Atlanta, Georgia, USA; Malaria Branch, Centers for Disease Control and Prevention, Atlanta, Georgia, USA; National Reference Laboratory, Rwanda Biomedical Centre, Kigali, Rwanda; US Peace Corps, Kigali, Rwanda; US President’s Malaria Initiative, Kigali, Rwanda; WHO Rwanda Office, Malaria and Neglected Tropical Diseases Programs, Kigali, Rwanda; The Johns Hopkins University, Bloomberg School of Public Health, Department of International Health, Baltimore, MD, USA