Preplanned Studies: Drug Resistance of Imported P. falciparum and P. vivax Isolates — China, 2021–2023

Introduction: Malaria remains the leading cause of infectious disease-related morbidity and mortality worldwide. ACTs continue to be the first-line treatment for uncomplicated malaria caused by P. falciparum. However, the global spread of antimalarial drug resistance, particularly artemisinin resistance, has become an increasing concern over time.

Methods: Therapeutic efficacy was evaluated following the World Health Organization’s guidelines for iDES. This study assessed adequate clinical and parasitological response (ACPR) and parasitemia on day 3 of treatment. Molecular surveillance of resistance-associated genes, including pfk13, pfcrt, and pfmdr1, was conducted on collected P. falciparum isolates.

Results: The iDES of AS plus DHA-PPQ was implemented in 2023, while CQ efficacy was monitored from 2021 to 2023. Late parasitological failure (LCF) for DHA-PPQ was detected in 1 of 26 P. falciparum cases in 2023, and in 1 of 26 and 1 of 90 P. vivax cases for CQ in 2022 and 2023, respectively. The corresponding ACPR rates were 96.2%, 100%, 96.2%, and 98.8%. The average positive parasitemia rate on day 3 post-treatment was 21.8%. Molecular polymorphism analysis revealed 9 nonsynonymous mutation haplotypes in the pfk13 gene, while 97.7% of samples presented the wild-type genotype. For the chloroquine resistance-associated pfcrt gene, 2 mutant haplotypes, ‘CVIET’ and ‘SVMNT’, were detected with frequencies of 16.7% (70/418) and 0.5% (2/418), respectively, while the wild-type haplotype ‘CVMNK’ predominated at 82.8% (346/418). In the pfmdr1 gene, 5 nonsynonymous point mutations and 8 haplotypes were identified. The Y184F mutation showed the highest prevalence at 40.5% (170/420). The 7 mutant haplotypes detected were V65L (0.2%, 1/420), N86Y (1.9%, 8/420), F408V (0.9%, 4/420), D1246Y (0.5%, 2/420), V65L/Y184F (0.2%, 1/420), N86Y/Y184F (0.7%, 3/420), and Y184F (39.8%, 167/420).

Conclusion: The antimalarial drug efficacy studies conducted for AS plus DHA-PPQ and CQ demonstrated that these treatments remain effective. However, the occurrence of LCF cases and persistent parasitemia on day 3 indicate decreasing sensitivity of these first-line drugs for treating P. falciparum and P. vivax, respectively. Therefore, continuous iDES and molecular surveillance of antimalarial drugs must be enhanced to provide early warning and guide appropriate responses to the spread of antimalarial drug resistance.

Malaria remains a significant global health challenge affecting 84 endemic countries, with an estimated 257 million infections and 597,000 deaths reported in 2023 (1). China achieved malaria-free certification from the World Health Organization (WHO) on June 30, 2021, following decades of elimination efforts (2). However, the increasing influx of Plasmodium-infected individuals from malaria-endemic regions, particularly sub-Saharan Africa and Southeast Asia, poses a substantial challenge to China’s post-elimination phase (3-4). Systematic monitoring of antimalarial drug efficacy aims to establish a centralized drug resistance database for imported strains, providing actionable intelligence for source countries and aligning with the WHO’s “High Burden to High Impact” strategy by informing region-specific antimalarial protocols (5).

Antimalarial drugs, especially artemisinin derivatives, are the most widely used treatments globally (6). Although China has reported no indigenous malaria cases since 2017, the challenges in preventing and controlling imported malaria have intensified, particularly with the emergence and spread of drug-resistant strains (6–7). While the treatment efficacy survey (TES) remains the gold standard for evaluating antimalarial drug efficacy, iDES are more appropriate in low-endemic or malaria elimination settings (5,8). This study employed an iDES protocol combined with analysis of drug resistance-associated molecular markers (pfk13, pfcrt, and pfmdr1) to monitor and evaluate the therapeutic efficacy of ACTs and CQ from 2021–2023, thereby providing baseline data to inform antimalarial drug policies.

The iDES and detection of drug resistance-associated molecular markers were performed according to previously described protocols (8–9). All experiments were conducted within the provincial malaria diagnostic laboratory network, with data uploaded to the National Information System for Parasitic Disease Prevention and Control. iDES was implemented to evaluate ACTs for P. falciparum treatment and CQ for P. vivax treatment in Yunnan Province. Patients with confirmed P. falciparum or P. vivax infection, excluding those with severe malaria, were followed from the first day of treatment (Day 0, D0) through D1-3, D7, D14, D21, D28, D35, and D42. Treatment outcomes were classified according to WHO guidelines for therapeutic efficacy monitoring as early treatment failure (ETF), LCF, late parasitological failure (LPF), or ACPR (5).

All malaria species were confirmed via PCR and microscopy. Molecular polymorphisms of PfK13, Pfcrt, and Pfmdr1 genes were detected and sequenced from imported malaria cases from 2021–2023. The chi-square test was used to evaluate differences in the distribution of drug resistance-associated gene polymorphisms, and data analysis was performed using GraphPad Prism 8.0 software.

A total of 174 cases of P. falciparum and P. vivax malaria were monitored for clinical efficacy through the iDES protocol (Table 1). In 2023, 26 cases of Pf malaria treated with AS combined with DHA-PPQ were included in the iDES follow-up. Among these, 25 patients achieved ACPR, while one patient tested positive for parasitemia on day 35, which was classified as late treatment failure. Additionally, 8 patients (30.8%, 8/26) exhibited persistent parasitemia on day 3 of treatment.

For Pv malaria, the treatment regimen consisted of chloroquine and primaquine. From 2021 to 2023, a total of 148 Pv malaria cases were followed up. In 2021, all 32 patients achieved ACPR, with only 1 patient showing parasitemia on day 3. In 2022, 4 out of 26 patients were lost to follow-up, 1 patient was classified as having late treatment failure (LTF), and 6 patients (27.2%, 6/22) demonstrated persistent parasitemia on day 3. In 2023, among the 90 cases targeted for follow-up, 8 were lost to follow-up, 1 case was identified as LTF, and 23 cases (28.0%, 23/82) presented with parasitemia on day 3.

A total of 451 cases of P. falciparum malaria were subjected to molecular testing for drug-resistance markers (Table 2). Of these, 442 samples were successfully sequenced for the pfk13 gene. 9 distinct point mutations were identified at the following loci: P441T, F446I, S459T, C469F, N499T, P574L, A578S, C580Y, and V692L (where the left side represents the wild-type allele and the right side represents the mutant allele). All the detected pfk13 mutations were single-point mutations. A total of 418 samples were successfully genotyped for the CQ resistance-associated pfcrt gene, with a focus on mutations at codons 72–76. 3 genotypes were identified (Table 3): the wild-type CVMNK and the mutant types CVIET and SVMNT. The most frequent mutation was K76T, which was detected in 72 samples. The SVMNT haplotype was observed in 2 samples, both originating from Southeast Asia (one from Indonesia and one from Bangladesh). In total, 420 samples were analyzed for pfmdr1 point mutations, revealing 5 mutation types and 7 mutant haplotypes. The most frequent mutation was Y184F, which was detected in 170 samples (40.5%), followed by N86Y (2.6%, 11/420). The 7 haplotypes included 2 novel combinations: l65f184 and Y₈₆F₁₄₈ (Table 3).

Regarding the clinical efficacy monitoring of AS plus DHA-PPQ, although this work was only conducted in 2023 due to policy requirements, the detection of parasitemia on day 3 (D3+) in 8 out of 26 cases clearly indicates prolonged parasite clearance time for ACTs, demonstrating decreased sensitivity to these drugs. Additionally, 1 patient tested positive for parasitemia by blood smear microscopy on day 35 (D35) posttreatment, which was classified as late treatment failure. Similarly, in the follow-up of P. vivax cases, the first parasite clearance time also showed an increasing trend over time. When excluding patients lost to follow-up, late treatment failure was observed each year. These phenomena suggest decreasing sensitivity of ACTs and CQ as first-line treatments for P. falciparum and P. vivax malaria, respectively. These findings further underscore the urgency and importance of the World Health Organization’s efforts to contain the spread of artemisinin resistance.

In the molecular detection of pfk13, this study identified K189T as a relatively dominant mutation site; however, it is not listed in Table 2 since it is not localized in the BTB/POZ region of the Kelch protein, the function of which remains unclear (10–11). This mutation was predominantly distributed in West African countries (such as Côte d’Ivoire, Nigeria, the Democratic Republic of the Congo, and Cameroon), with 1 case each detected in North Africa and East Africa (from Zambia and Algeria, respectively). Other newly detected mutation sites including S459T, N499T, A578S, and V692L were reported for the first time (10). However, it remains unclear whether these novel mutations will become fixed over time. For the pfcrt gene, the K76T point mutation is highly associated with CQ resistance. Nevertheless, the proportion of the wild-type genotype exceeded 82%, and compared with the variation in pfmdr1, the difference between pfcrt and pfmdr1 was statistically significant by Chi-square test (P<0.01), supporting the observation that sensitivity to CQ is gradually recovering following the withdrawal of CQ (12–13). Furthermore, detection of pfmdr1 gene mutations revealed that Y184F was the most frequently mutated site, indicating its potential association with multidrug resistance, particularly in the context of resistance to ACTs and their partner drugs.

In conclusion, this study employed two approaches — iDES and drug resistance-associated molecular detection — to evaluate the efficacy of antimalarial drugs. As the current gold standard for clinical drug efficacy evaluation, iDES requires sustained and substantial investments in human and material resources. Based on the initial results presented in this study, future efforts must leverage the comprehensive parasitic disease prevention and management information system to achieve two critical objectives: 1) expanding the clinical follow-up sample size and 2) ensuring nationwide coverage of imported malaria cases. These steps will enable more objective and timely assessments of antimalarial drug efficacy in real-time settings, thereby mitigating the risk of further drug resistance dissemination.

The staff involved in field investigations and to provincial CDC personnel participating in the national antimalarial drug resistance network. We also thank all malaria patients who were recruited for the iDES.