Malaria poses a significant threat to global public health. According to the World Malaria Report, 241 million clinical cases and 627,000 malaria-related deaths were reported worldwide in 2020 (1). Plasmodium falciparum is the most prevalent and virulent malaria species, causing approximately 90% of malaria-related deaths each year. The World Health Organization (WHO) African region accounts for 95% of malaria cases globally (1, 2). Malaria in Africa is also a continuing risk for international travelers, especially long-term migrant workers (2). Anti-malarials are the main weapon used against this potentially lethal infection (3). Despite significant progress in the fight against malaria worldwide, the spread of anti-malarial resistance threatens the current efforts to control malaria in Africa, as well as in countries where malaria cases have been imported (3, 4). China was certified as malaria-free by the WHO in June 2021 (5); however, the disease still poses a therapeutic challenge due to imported malaria cases.

Artemisinin resistance is primarily mediated by mutations in P. falciparum Kelch13 protein (K13), which involved in intracellular processes, such as endocytosis of hemoglobin (6). To date, a list of 10 validated P. falciparum k13 markers and 11 candidate markers have been used to evaluate partial artemisinin (ART) resistance (7). It was thought that the validated k13 markers were mainly limited to Southeast Asia until a validated marker, R561H, was identified in 7.4% of samples (19 of 257) between 2013 and 2015 in Rwanda, which had no genetic relationship with those previously reported in Southeast Asia. This finding has led to great concern given the disease burden in Africa (8). Pfcrt, which encodes the P. falciparum chloroquine resistance transporter, is a highly sensitive marker of chloroquine (CQ) treatment failure (9). The Pfcrt gene has a considerable polymorphism in codon position 72–76 that determines different haplotypes, including wild-type CVMNK, common mutation CVIET, SVMNT and mixed mutation type. The P. falciparum multidrug resistance protein transporter is a central node in a wide range of anti-malarial resistance through single nucleotide polymorphisms and/or copy numbers in Pfmdr1 (10, 11). Pfmdr1 is associated with parasite susceptibility to a variety of currently available anti-malarial drugs, including CQ, ART, lumefantrine, amodiaquine, mefloquine, and quinine (10). Most of them are important counterparts in first-line Artemisinin-based Combination Therapy (ACT); thus, Pfmdr1 has become one of the pivotal factors in malaria resistance to artemisinin combination therapies (10, 11).

By subregion, central African countries are malaria endemic and accounted for nearly 25% of the estimated malaria cases in the WHO African region in 2020, which includes 10 countries of Congo DR, Angola, Cameroon, Equatorial Guinea, Gabon, Congo, Chad, Central African Republic, Sao Tome and Principe, and Burundi (1). A large number of malaria infections are imported from central Africa to China each year, particularly from Congo, Angola, Cameroon, etc. (12, 13). This poses a great risk to China in maintaining a malaria-free status, as imported patients with anti-malarial drug resistance might result in failure or delay in parasite clearance (14, 15). Therefore, it is necessary to follow the epidemiological patterns of imported malaria infections that are resistant to malaria drugs. Herein, we examined the prevalence of the molecular markers of k13, pfcrt, and pfmdr1, and their polymorphisms in imported P. falciparum malaria cases from central Africa between 2016 and 2021 in Zhejiang province, China. This study aimed to provide useful information for a deeper understanding of the status and spectrum of drug resistance of the P. falciparum parasite in central Africa by examining the molecular epidemiology traits of imported cases, and to gain information that can be used to provide guidance on medication in clinical practice and drug administration.

It is essential that anti-malarial drugs remain effective in saving the lives of millions of people infected with malaria. Monitoring drug resistance is important because early detection enables a timely action in order to prevent its spread and to mitigate any impact on global health. Most of the global burden of P. falciparum is in the WHO African region. In 2020, it was estimated that there were more than 54 million infections and almost 140,100 deaths in central Africa and approximately 191 million people living in the 10 countries of central Africa are at high risk of malaria (1). Here, we investigated the genetic mutations of k13, Pfcrt, and Pfmdr1 associated with anti-malarial resistance in malaria cases imported from central Africa to Zhejiang province, China. Our purpose was to better understand the status and spectrum of drug resistance of parasites in central Africa using molecular epidemiology and to provide evidence for drug policy updates in the study area.

The present observational study illustrated a low prevalence of nonsynonymous mutations of k13 (0.9%, 2/232) in the samples analyzed, indicating limited k13 polymorphism in central African countries. This finding is comparable with the available data (19–22). A review by the WHO indicated that most of the samples in central African countries were k13 wild type (98.6%) (23). In summary, 61 mutations were identified, of which five (M476I, P574L, P553L, R539T, and R561H) were validated molecular markers and one (C469F) was a candidate molecular marker associated with partial ART resistance (23). Notably, neither validated markers nor candidate markers were identified in our study. Regarding the two non-synonymous mutations (D464E and K503E) revealed in this study, there is still no evidence confirming their roles in mutant k13-mediated ART resistance. In addition to the validated and candidate molecules, the rare frequency of some nonsynonymous mutations, such as D464E, K503E, and I448L in Cameroon, V454A in Gabon, and V534A in Congo, described in previous studies, underlines the abundant polymorphism of k13 in central Africa (24). At the country level, Cameroon presented more k13 polymorphisms than any other central African country, with one sample possessing nonsynonymous mutations, and three samples possessing synonymous mutations. Although this difference may be due to more participants being enrolled from Cameroon in the current study, continued monitoring of the trend of molecular determinants is warranted, given the genetically independent rise of R561H in African isolates since 2014 (8).

It was demonstrated that the mutant haplotype CVIET in Pfcrt conferred a high level of resistance and was the most common in Africa (25). Our data demonstrated a low proportion (19.5%, 39/223) of mutant type CVIET in Pfcrt at positions 72–76, indicating the return of CQ-sensitive P. falciparum parasites in central Africa. This was consistent with previous studies, which revealed a prevalence of more than 90% prior to CQ withdrawal in 2002, which declined to 73.2% 8 years after CQ replacement in Congo (26, 27), as well as the reemergence of P. falciparum isolates with wild-type Pfcrt in Cameroon, Angola, and Equatorial Guinea (22, 28, 29). Based on our findings, it is suggested that the selection of Pfcrt was relieved in most central African countries after long-term ACT use. However, Gabon was underscored in the present study because of its low prevalence of wild-type CVMNK (30.8%) compared with other countries from central Africa. Although the current study was limited by the small size of Gabon, the results were in agreement with those of another study, which showed a low wild-type proportion of 16.5–29.4% in Gabon (30).

Combinations of SNPs in codons N86Y, Y184F, and D1246Y of Pfmdr1 confer reduced sensitivity to different drugs. Parasites with wild N86 was estimated 4.7 times more likely to recrudesce after Artemether–lumefantrine treatment than those with the N86Y genotype (31, 32). Concerning the polymorphism of Pfmdr1, we found that Y184F was the predominant mutation with a prevalence of 61.1%, which was much higher than those that identified in central Africa, as well as east and west Africa, in other published studies (33, 34). Furthermore, more than half of the samples from central Africa in the current study harbored a single mutant haplotype NF, suggesting that NF was more common in central Africa than previously thought (35). In addition, the frequency of haplotypes of Pfmdr1 varied greatly between countries. The single mutant haplotype NF was more frequently observed in Congo (69.2%), Gabon (56.3%), and Equatorial Guinea (59.5%) when compared to other countries, indicating the diversity of drug pressure and transmission intensity among African countries or regions.

Regarding the temporal distribution of haplotypes of Pfcrt and Pfmdr1, our data indicated a return of sensitive Pfcrt and Pfmdr1. Specifically, there was an increase in the prevalence of wild-type CVMNK from 68.5–74.6% in 2016–2017 to 81.8–87.5% in 2018–2021. For Pfmdr1, an increasing proportion of wild-type NY and declining frequency of mutant type were demonstrated between 2016 and 2019. The moderate rise of wild-type Pfcrt and Pfmdr1 in the present study indicated the relief of drug pressure due to the cessation of CQ use in central African countries (27, 28). Therapeutic efficacy studies in central Africa are recommended for tracking clinical and parasitological outcomes (36).

This study had some limitations. First, there was a small sample size in several countries, especially the northern countries of central Africa, which limited further interpretations of the prevalence of molecular markers. However, the geographic distribution of the parasites in the present study was similar to that of malaria cases in central African countries. Congo, Angola, and Cameroon account for approximately 80% of the estimated infections (1), and the imported P. falciparum cases in our study were mainly from these three countries. Second, no data on the success or clinical failure of the treatments were collected, as the current study focused on the molecular markers associated with drug resistance in P. falciparum parasites.

In this study, we demonstrated the geographical and temporal distribution of k13 propeller, Pfcrt and Pfmdr1 polymorphisms in P. falciparum parasites imported from central Africa between 2016 and 2021. A limited k13 propeller polymorphism was identified. The relief of antimalarial drug pressure in central Africa was indicated by predominant wild type CVMNK of Pfcrt and the single mutant of Pfmdr1 in most countries, as well as their increasing trend during the study period between 2016 and 2021. The profiles of haplotypes of Pfcrt and Pfmdr1 displayed varied spatial patterns among countries. Especially, Gabon and Equatorial Guinea required further strategy and measures as their higher proportion of mutant/mixed type of Pfcrt or Pfmdr1, comparing with other countries. Our data provides updated evidence that can be used to adjust national anti-malarial drug policies in central Africa and China.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors.

The studies involving humans were approved by Ethical Review Committee of Zhejiang Center for Disease Control and Prevention. The studies were conducted in accordance with the local legislation and institutional requirements. The ethics committee/institutional review board waived the requirement of written informed consent for participation from the participants or the participants’ legal guardians/next of kin because Malaria is the notificable infectious in China, and the epidemiological investigation of notificable infectious cases are conducted in accordance with the Infectious Disease Prevention and Control Law.

XW and WR conceived this study. HC, JZ, and QL carried out the molecular studies. XZ analyzed the data. WX and XW drafted the manuscript. All authors contributed to the article and approved the submitted version.

This work was funded by grants from the Basic public welfare research project of Zhejiang Province (Grant No. LGF22H260006) and Medical Research Program of Zhejiang Province (Grant No. 2021KY121 and 2022KY723).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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