Targeted deep amplicon sequencing of kelch 13 and cytochrome b in Plasmodium falciparum isolates from an endemic African country using the Malaria Resistance Surveillance (MaRS) protocol

Abstract

Background: Routine molecular surveillance for imported drug-resistant malaria parasites to the USA and European Union is an important public health activity. The obtained molecular data are used to help keep chemoprophylaxis and treatment guidelines up to date for persons traveling to malaria endemic countries. Recent advances in next-generation sequencing (NGS) technologies provide a new and effective way of tracking malaria drug-resistant parasites. Methods: As part of a technology transfer arrangement between the CDC Malaria Branch and the Istituto Superiore di Sanità (ISS), Rome, Italy, the recently described Malaria Resistance Surveillance (MaRS) protocol was used to genotype 148 Plasmodium falciparum isolates from Eritrea for kelch 13 (k13) and cytochrome b (cytb) genes, molecular markers associated with resistance to artemisinin (ART) and atovaquone/proguanil (AP), respectively. Results: Spanning the full-length k13 gene, seven non-synonymous single nucleotide polymorphisms (SNPs) were found (K189N, K189T, E208K, D281V, E401Q, R622I and T535M), of which none have been associated with artemisinin resistance. No mutations were found in cytochrome b. Conclusion: All patients successfully genotyped carried parasites susceptible to ART and AP treatment. Future studies between CDC Malaria Branch and ISS are planned to expand the MaRS system, including data sharing, in an effort to maintain up to date treatment guidelines for travelers to malaria endemic countries.

Antimalarial Drug Resistance and Implications for the WHO Global Technical Strategy

“…Next-generation sequencing (NGS) methods, such as targeted amplicon deep sequencing (TADS), are increasingly used to monitor molecular markers of resistance [174][175][176][177][178]. By enabling high-sensitivity, high-throughput analysis of molecular markers, the declining costs and increasing data yield of NGS have positioned it to replace traditional PCR-based methods [179].…”

Prevalence of potential mediators of artemisinin resistance in African isolates of Plasmodium falciparum

“…The reported pfk13 non-synonymous SNPs occurring inside the propeller domain (amino acid from 443) include A578S/D/V (95 parasite isolates with the SNP); R561H (20 isolates with the SNP); R622G/K/I (20 isolates with SNP); N587K/I (16 isolates); V555A/L (9 isolates); S522C/M/N (9 isolates); T677A/K/R (9 isolates); Q613E/H (7 isolates), F509G (7 isolates) and V637I (6 isolates); N554H/K/D and A626S/T/V (5 isolates each); and, N609D/L/S (5 isolates) [35,[55][56][57][58]. The most frequently reported mutations outside the propeller domain (amino acid below 443) include K189T/N (105 isolates with the mutation) [51,59], E208K (10 isolates) [60], N142NN (9 isolates) [61], T149S (6 isolates) [62], E433D (4 isolates) [54], and E401Q. Apart from D389H/N/Y (3 isolates), K378R (2 isolates) and D281V (2 isolates), other reported mutations outside the propeller domain (31/40) occurred singly [54,59].…”

Mutation in the Plasmodium falciparum BTB/POZ Domain of K13 Protein Confers Artemisinin Resistance

“…Remarkably, the P413A mutation is located in the BTB/POZ domain of PfK13 (amino acids 350–437) ( 16 ). To date, only 17 non-synonymous mutations located in the BTB/POZ domain of PfK13 have been reported in clinical isolates and only one, D353Y (found in five isolates from Vietnam), was associated with a parasite clearance half-life (PCt 1/2 ) > 5 h after ACT treatment (Table S7) ( 23 , 24 , 33 – 40 ). Neither P413A nor other missense mutations at position 413 have been reported so far, whereas the synonymous mutation P413P (codon CCC → CCG or CCT) was reported in 12 African and Asian isolates ( 23 ).…”