Background

Artemisinin-based Combination Therapy (ACT), a key pillar in the global fight against malaria, is facing intense resistance in Africa. ACT, prescribed for Plasmodium falciparum infections, pairs artemisinin derivatives, which rapidly kill parasites, with partner drugs to eliminate remaining parasites. Over the past few decades, this combination therapy has been regarded as a key shield that has significantly contributed to reducing malaria mortality.

The problem is that artemisinin partial resistance, which began in Southeast Asia, has recently spread to East Africa. Health authorities have used mutations in the Kelch13 (k13) gene on chromosome 13 as the primary indicator for tracking resistance. However, in clinical practice, there have been frequent cases of delayed treatment response and drug resistance that cannot be explained by K13 mutations alone. It was time to identify unknown genetic factors that inhibit the efficacy of not only artemisinin but also partner drugs. This was the background against which the existing surveillance network, centered on single markers, revealed limitations in capturing the risk of complex protozoan resistance.

Key Findings

An international research team, through precise analysis of patient samples from Uganda, identified the key genetic variants driving drug resistance. Analysis of whole-genome sequencing (WGS) data from 157 Plasmodium falciparum specimens collected in Uganda revealed a strong selective sweep centered on the pfcrt gene on chromosome 7, in addition to k13 on chromosome 13. This is described as a genomic signal observed when genes advantageous for survival spread rapidly within a population.

This region formed a single haplotype combining three non-synonymous missense variants and two microdeletions. The researchers named this combination of genetic variants the 'PIN' haplotype. Genotyping 1,598 specimens collected in eastern and northern Uganda over 20 years, from 2004 to 2024, confirmed that the PIN haplotype has gradually increased in frequency since its first discovery in 2008. It has now established itself as the dominant genotype in Plasmodium falciparum populations across Uganda.

Laboratory validation also clearly confirmed actual drug resistance. As a result of ex vivo drug susceptibility evaluation of cultured patient-derived parasites, PIN haplotype parasites showed a significant decrease in dihydroartemisinin (DHA) susceptibility. The same phenomenon was observed in knockout parasite clones where the px1 gene was deleted. Results also showed a significant decrease in susceptibility to lumefantrine and mefloquine, the primary partner drugs used in patient treatment.

Implications and Outlook

This study calls for a complete revision of malaria prevention strategies. This is because it has been proven that a multidrug-resistant genotype, which neutralizes partner drugs alongside decreased artemisinin sensitivity, has already become established at the population level. Analysis suggests that existing diagnostic methods tracking only the K13 gene will struggle to detect the spread of resistance in sub-Saharan Africa at an early stage.

Public health authorities in each country must urgently incorporate the px1 gene and PIN haplotype into molecular diagnostic surveillance systems. The pharmaceutical industry also needs to accelerate the expansion of new drug pipelines with novel mechanisms of action. There is growing concern that the effective lifespan of existing combination therapies may be shorter than expected.

However, subsequent verification challenges remain evident. The exact molecular biological function of the PX1 protein and the pathway inducing drug resistance have not yet been clearly elucidated. There is a pressing need for large-scale prospective studies to determine how significantly the final treatment failure rate rises among patients infected with the PIN haplotype in actual clinical settings.