We attempted Pfk13 sequencing on 3,248 samples (2,579 pretreatment and 669 day of failure samples) from the 9 countries (Table 2); 2,865 were successfully sequenced (Table 3). Of those, 2,753 samples were wild-type. A total of 11 unique nonsynonymous mutations and 27 unique synonymous mutations were detected in 2,865 successfully sequenced pretreatment and day of failure samples from Angola, Benin, DRC, Guinea, Kenya, Malawi, Mali, Tanzania, and Zambia collected during 2014–2018 (Figure, Table 4; Appendix 1).

Of the 2,303 sequenced pretreatment samples, 2,213 were wild-type and 90 (3.9%) contained mutations (Table 3). Of the 90 pretreatment samples with mutations, 10 unique nonsynonymous mutations were present in 25 samples from 8 of the 9 countries assessed (Table 4) and 25 unique synonymous mutations were present in 65 samples from 8 of the 9 countries assessed (Appendix 1 Table 1). Two samples from Kenya contained the S522C mutation, reported by WHO as a less-frequent mutation associated with delayed parasite clearance but without statistical significance because of limited data (3). Both of these patients cleared their initial infection. A578S, the most commonly found mutation in Africa (not associated with resistance) (3), was the most common nonsynonymous mutation we identified. The mutation was found in 14 pretreatment isolates: 4 in Angola, 1 in DRC, 1 in Mali, 6 in Kenya, 1 in Tanzania, and 1 in Zambia (Table 4). No mutations were identified in the samples from Malawi. Eight of the 10 unique nonsynonymous mutations in the pretreatment samples have been reported previously in other countries, whereas 2 mutations, P419S (Guinea) and Q613R (Angola), were newly identified in our study. No WHO-validated or candidate Pfk13 mutations were identified.

Of the 669 day of failure samples, 562 were successfully sequenced; 107 (16.0%) samples failed to amplify, produced poor-quality sequences, or both (Table 3). A total of 540 samples were wild-type. Two nonsynonymous mutations were found in 4 day of failure samples (Table 4) and 10 synonymous mutations (Appendix 1 Table 2) were identified in 18 day of failure samples from 4 countries. Of the nonsynonymous mutations in day of failure samples, 2 samples from Kenya and 1 sample from DRC contained the A578S mutation, and 1 sample from DRC contained the S477Y mutation (Table 4). We compiled the complete results of the sequence data reanalysis (Appendix 2).

This work provides an update on Pfk13 genetic markers in 9 countries in Africa with endemic malaria. Although clinical resistance to ACTs has yet to be confirmed in Africa (1), the early detection of Pfk13 mutations through surveillance allows for swift action before resistance spreads widely. To date, all WHO-validated SNPs detected in Africa have been the result of independent emergence as opposed to spreading through imported cases from Southeast Asia (21). More than 200 Pfk13 mutations have been identified in global samples (3,18,21), and >74 Pfk13 nonsynonymous mutations have been reported in Africa (22,23). In this study, we report the presence of S522C in Kenya, a less frequent mutation that has been previously reported to be associated with delayed parasite clearance but lacking sufficient evidence to be considered a WHO-validated or candidate mutation (3).

As more molecular surveillance data are collected, previous results should be reinterpreted to determine the presence of WHO-reportable mutations because the importance of these mutations in drug resistance might change based on new data (3,24). Although we report only 1 mutation identified by WHO to possibly play a role in resistance, other detected mutations, such as the other nonsynonymous mutations with unknown resistance status reported in this study, might be deemed important in the future as more data are collected and validated. In 2017, WHO categorized only 5 mutations as validated (N458Y, Y493H, R539T, I543T, and 580Y) (24), but in 2018 the validated list was updated to include an additional 4 mutations, including F446I, P553L, and R561H (formerly candidate markers) and M476I (formerly reported as a less frequent variant associated with in vivo or in vitro test results) (3). In addition, the Worldwide Antimalarial Resistance Network tracks Pfk13 mutations worldwide and strives to detect new associations of mutations with delayed parasite clearance, which might inform WHO classifications (18).

We report the presence of 11 unique nonsynonymous mutations in Angola, Benin, Guinea, DRC, Kenya, Mali, Tanzania, and Zambia; all were previously reported in the literature (Table 4) except P419S and Q613R. The most common nonsynonymous mutation observed in our study was A578S, a nonsynonymous mutation frequently described in Africa (3) and, to a lesser extent, Asia (e.g., Thailand [19] and Bangladesh [25]). WHO has reported that A578S is not associated with partial artemisinin resistance (3). Most mutations detected were synonymous mutations consistent with previous reports (21). Because synonymous mutations do not result in an amino acid change, they are not associated with resistance. Parasites from Africa have been shown to have a higher prevalence of synonymous mutations, which is not surprising given that P. falciparum originated in Africa and continues to have a high level of transmission in this region (19).

The results described in this article represent the collaborative output of the PARMA network, which originated in 2015 with the objectives of assisting countries in Africa in testing malaria samples from TESs for genetic markers associated with antimalarial resistance and supporting training and capacity building of collaborators in Africa (9). In 8 of the 9 countries included in this report (all but Angola), the Pfk13 results were generated during a 6–8-week visit to CDC by trainees from a laboratory in the country where the TES was performed. Results were subsequently shared by the trainee’s laboratory with their national malaria control program and other local stakeholders to make decisions related to antimalarial use. Although the Pfk13 results we have described would not be cause for alarm or policy change, recent findings in Rwanda suggests a substantial presence of the Pfk13 R561H mutation (26) that has evolved locally, highlighting the importance of molecular surveillance for early detection of emerging patterns of resistance. In this context, PARMA training visits generate a vast amount of data from TES samples, ranging from efficacy results to prevalence of other molecular markers (e.g., P. falciparum multidrug-resistant protein 1 and P. falciparum chloroquine-resistance transporter) to the presence of P. falciparum histidine-rich protein 2 and 3 deletions (which might affect rapid diagnostic test performance). Generating phenotypic (i.e., efficacy) and genotypic data on the same sample provides an opportunity to identify novel mutations associated with resistance and enables detection of known mutations in samples with well-characterized efficacy outcomes. Because the PARMA network encourages standardization of laboratory methods and data reporting, such explorations might detect trends over time in a single country or produce insightful observations by using data from multiple countries. With the increased use of next-generation sequencing, the PARMA network has embarked on applying these principles of data generation, capacity building, networking, and standardization to this emerging technology (27). The ultimate goal is laboratories in Africa independently analyzing their own malaria samples.