We conducted a hospital-based cross-sectional observational study during August–December 2017 in Al Jazirah and Al Qadarif states, located in the east-central region of Sudan. Those sites are characterized by a subequatorial climate with a rainy season typically occurring during June–early November (13) (Appendix). All patients exhibiting clinical symptoms suggestive of malaria were eligible for blood sampling, after informed consent was obtained from the patient or parents. We confirmed P. falciparum malaria diagnosis through microscopy by using thick and thin blood films. Patients received treatment with AL drugs in accordance with the national drug policy. We spotted finger-prick blood onto Whatman 3MM filter papers (Whatman International, https://www.cytivalifesciences.com) to make dried blood spots for molecular analysis. The study received ethical approval from the Ministry of Health in Gezira State (approval no. MU/2019).

We extracted genomic DNA from three 3-mm punches of dried blood spots, as previously described (14). We conducted Illumina paired-end sequencing (Illumina, https://www.illumina.com) and selective amplification of parasite DNA to identify single-nucleotide polymorphisms in the Pfkelch13, Pfcrt, Pfmdr-1, dhfr, and dhps genes, as previously described (8). In addition, we assessed deletions of the hrp2 or hrp3 genes, which can lead to false-negative results in HRP2-based RDTs. We used laboratory reference parasite strains (Dd2, 7G8, HB3, and Cambodia culture-adapted strains) with known alleles and the presence or absence of hrp2 and hrp3 deletions as controls (Appendix).

We collected a total of 257 blood samples from 2 study sites: 170 from Al Jazirah and 87 from Al Qadarif. Demographic data were missing for 2 samples, resulting in a study population of 255 participants, 128 female and 127 male, ages 1–44 years. Most enrolled patients were febrile (95.6%), and their clinical manifestations were common to malaria, such as headache and vomiting (58.6%). After we examined the thick and thin blood films, we found all the patients were positive for P. falciparum.

Molecular analysis of the 257 samples revealed a high prevalence of the Pfkelch13 622I mutant parasites (21.8%, n = 56), a mutation validated by the World Health Organization as associated with ART-R. Among them, we identified 192 Pfkelch13 wild-type, 52 Pfkelch13 622I single mutants, 1 Pfkelch13 494I single mutant, 7 Pfkelch13 625R single mutants, and 5 isolates with polyclonal infections (3 Pfkelch13 622I/625R, 1 Pfkelch13 622I/494I, and Pfkelch13 494I/658T) (Table 1).

We further explored the genetic profile of the Pfkelch13 R622 wild-type parasites from Sudan and 622I mutants at known antimalarial drug–resistance loci and the frequency of hrp2 and hrp3 deletions, a genomic feature previously observed in Pfkelch13 622I mutants in Eritrea and Ethiopia (8,10). We examined 201 Pfkelch13 R622 wild-type and 56 Pfkelch13 622I mutant parasites for mutations across 4 genes (Table 2). Differences in allele frequencies were noted in the Pfcrt gene, associated with resistance to chloroquine, and the dhfr gene, associated with resistance to pyrimethamine. We conducted a stratified analysis that revealed a higher difference in Pfcrt allele frequencies in the Al Qadarif site compared with Al Jazirah. High proportions of Pfkelch13 622I parasites exhibited Pfcrt 74I/75E/76T/356T mutations (5.4% vs. 0% for Pfkelch13 R622 wild-type parasites; p = 0.01) as well as the dhfr wild-type allele (41.1% vs. 18.9% for Pfkelch13 R622 wild-type parasites; p = 0.001).

Figure

Figure. Distribution of artemisinin-resistant Plasmodium falciparum, southeastern Sudan, 2017. Percentages of Pfkelch13 R622 and Pfkelch13 622I P. falciparum parasites with no hrp2/3...

We also analyzed hrp2 and hrp3 deletions. We found a higher proportion of Pfkelch13 622I parasites with both hrp2 and hrp3 deletions, compared with Pfkelch13 R622 wild-type parasites (10.7% vs. 3.0%; p = 0.02) (Table 2; Figure). This difference was significant in samples from Al Jazirah (p = 0.04).

The spread of the Pfkelch13 622I mutant in the Horn of Africa is alarming because it raises the potential for ART-R to take hold in this region. Data from this study show parallels with findings from Eritrea (8) and possibly Ethiopia (10). With confirmed cases in Eritrea and possible cases in Ethiopia (8,10), the presence of this mutant in Sudan suggests a possible cross-border spread that could threaten malaria treatment in several countries. If left uncontained, Pfkelch13 622I mutant parasites could undermine the efficacy of ACTs, threatening progress in malaria control in East Africa. Rapid regional surveillance and coordinated response efforts are urgently needed to contain this threat before it escalates further. In addition, the presence of hrp2/hrp3 gene deletions in 10.7% of the Pfkelch13 622I mutants reported in this study is an additional concern, because it affects the results of HRP2-based RDTs in detecting P. falciparum parasites. Because of the presence of those gene deletions, the variants could spread more easily because they are not detected by the HRP2-based RDT, complicating malaria control efforts in affected regions.

The first limitation of this study is that we could not confirm the association between the presence of Pfkelch13 622I and the persistence of parasitemia on day 3, because clinical data were not available. Second, our data came from only 2 health facilities with limited catchment areas. Third, the samples were collected in 2017, but the findings remain highly relevant in 2025 because they provide critical baseline data on the early spread of resistance markers in Sudan. Although clinical reports of treatment failures in Sudan remain limited, our data are essential for understanding the persistence and regional transmission dynamics of ART-R P. falciparum parasites, which is crucial for guiding current malaria control strategies.

Our findings increase the need for further studies to determine whether the Pfkelch13 622I mutants observed in Sudan represent a local emergence or are directly linked to the P. falciparum parasite population from Eritrea or Ethiopia, as well as to clarify the flow of the Pfkelch13 622I mutant parasite population in this region. With ART-R now confirmed in P. falciparum and hrp2 and hrp3 deletions in parasite populations in Sudan, additional strategies must be implemented to contain the spread of these lineages across the Horn of Africa. Otherwise, the emergence of partner drug resistance could lead to higher rates of treatment failure and uncontrolled spread of potentially resistant P. falciparum parasites beyond this region.