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International Journal for Parasitology: Drugs and Drug Resistance logoLink to International Journal for Parasitology: Drugs and Drug Resistance
. 2024 Mar 19;24:100532. doi: 10.1016/j.ijpddr.2024.100532

Absence of association between Pfnfs1 mutation and in vitro susceptibility to lumefantrine in Plasmodium falciparum

Weilin Zeng a,1, Wei Zhao a,1, Hao Wei b, Yucheng Qin b, Zheng Xiang a, Yanrui Wu a, Xi Chen a, Yanmei Zhang a, Hui Zhao a, Mengxi Duan a, Wenya Zhu a, Kemin Sun a, Yiman Wu a, Tao Liang a, Ye Mou a, Cheng Liu a, Xiuya Tang a, Yaming Huang c, Liwang Cui d,⁎, Zhaoqing Yang a,⁎⁎
PMCID: PMC10979268  PMID: 38520842

Abstract

Artemether-lumefantrine (AL) is the most widely used antimalarial drug for treating uncomplicated falciparum malaria. This study evaluated whether the K65Q mutation in the Plasmodium falciparum cysteine desulfurase IscS (Pfnfs1) gene was associated with alternated susceptibility to lumefantrine using clinical parasite samples from Ghana and the China-Myanmar border area. Parasite isolates from the China-Myanmar border had significantly higher IC50 values to lumefantrine than parasites from Ghana. In addition, the K65 allele was significantly more prevalent in the Ghanaian parasites (34.5%) than in the China-Myanmar border samples (6.8%). However, no difference was observed in the lumefantrine IC50 value between the Pfnfs1 reference K65 allele and the non reference 65Q allele in parasites from the two regions. These data suggest that the Pfnfs1 K65Q mutation may not be a reliable marker for reduced susceptibility to lumefantrine.

Keywords: Plasmodium falciparum, In vitro susceptibility, Lumefantrine, Pfnfs1 gene

Graphical abstract

Image 1

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1. Introduction

According to the 2023 report from the World Health Organization (WHO), there were 249 million malaria cases and 608,000 deaths worldwide in 2022 (WHO, 2023), with Africa accounting for approximately 95% of global cases. Plasmodium falciparum is the most prevalent species in Africa, whereas in Southeast Asia, such as countries in the Greater Mekong Subregion (GMS), P. falciparum is on the verge of elimination.

Chemotherapy is an essential tool for malaria control. Due to the widespread resistance to chloroquine (CQ) and later to the antifolate drug sulfadoxine-pyrimethamine (SP), artemisinin-based combination treatments (ACTs) were adopted worldwide as the first-line treatment for uncomplicated P. falciparum malaria in the early 21st century (Ashley and White, 2005). However, the emergence of ART resistance in the GMS of Southeast Asia a decade ago (Amaratunga et al., 2012; Ashley et al., 2014; Dondorp et al., 2009; Noedl et al., 2008) and recently in East Africa (Balikagala et al., 2021; Straimer et al., 2022; Uwimana et al., 2020, 2021) is a serious concern. In addition, an increasing number of studies have reported the failure of ACT treatment among travelers returning to non-endemic countries (Grossman et al., 2023; Pierreux et al., 2023; Rovira-Vallbona et al., 2019; Silva-Pinto et al., 2021).

Artemisinin partial resistance is mainly correlated with mutations in the kelch13 (PfK13) gene of P. falciparum (Ariey et al., 2014; Ocan et al., 2019). For the ACT partner drugs, mutations in the P. falciparum chloroquine resistance transporter (PfCRT) and P. falciparum multidrug resistance 1 transporter (PfMDR1) confer resistance to 4-aminoquinoline drugs such as CQ and amodiaquine (Ross and Fidock, 2019), while piperaquine (PQ) resistance is due to plasmepsin II and III amplification (Amato et al., 2017) and PfCRT mutations (Ross et al., 2018). The mode of action of aryl-alcohol drugs, such as lumefantrine (LMF), is not yet clear, but it is widely believed that these drugs can interfere with heme detoxification (Wicht et al., 2020). A recent population genomic study comparing temporal population differentiation with parasite samples collected from The Gambia identified a non-synonymous mutation, K65Q, in the cysteine desulfurase IscS (Pfnfs1) (PF3D7_0727200) gene, which had undergone strong directional selection (Amambua-Ngwa et al., 2018). Along with the extensive deployment of artemether-lumefantrine (AL), the reference allele frequency had increased from 10% in 2008 to 32% in 2015, suggesting selection by this ACT. The reference allele was also associated with in vitro resistance to LMF (Amambua-Ngwa et al., 2018). In western Kenya, the frequency of the reference K65 allele was found to be higher in patients with recurrent infection after ACT treatments (Gachie et al., 2023). Therefore, monitoring molecular markers related to drug resistance in ACTs is crucial for early detection and response to emerging drug resistance.

Ghana in West Africa is among the malaria high-burden countries, with over 5 million malaria cases in 2021 (WHO, 2023). In 2005, Ghana introduced artesunate-amodiaquine (AS-AQ) as the first-line treatment, followed by the introduction of AL and dihydroartemisinin-piperaquine (DHA-PPQ) in 2008 (Abuaku et al., 2012). In recent years, Ghana has been one of the main sources of imported malaria cases in China. In 2013 and 2016–2017, Ghana accounted for 99.7% and 58% of imported malaria cases in Shanglin County, Guangxi, respectively (Li et al., 2015; Liu et al., 2021). The China-Myanmar border was once a high-risk area for malaria, with a unique history of antimalarial drug use. LMF was used in the China-Myanmar border area as early as the 1990s (Che et al., 1994a, 1994b). In addition, artemisinin-based drugs were mainly used as monotherapies before 2005. After 2005, the national antimalaria drug policy adopted ACT, mainly DHA-PPQ, as the first-line treatment for uncomplicated cases of P. falciparum in the region.

With the finding of elevated resistance of parasites from East Africa to LMF and the potential selection of Pfnfs1 in West Africa, we wanted to evaluate the prevalence of Pfnfs1 mutation in isolates from Ghana and the China-Myanmar border area, and then investigate its relationship with the in vitro susceptibility of the parasite to LMF.

2. Materials and methods

Ethical statement

All patients in this study voluntarily signed informed consent forms, and the research project was approved by the Institutional Review Committee of Kunming Medical University.

2.1. Parasite isolates collection

The Ghanaian isolates were collected from Chinese migrant workers who had returned from Ghana. These patients with malaria symptoms attended the Guangxi Shanglin Hospital from 2016 to 2018 and were diagnosed with malaria by microscopy using Giemsa-stained thick and thin blood smears. Those with complex travel histories and those who used antimalarial drugs within the previous month were excluded from the study. The isolates from the China-Myanmar border were archived clinical samples collected from the Nabang township in west Yunnan Province, China, and the Laiza township, Kachin State, Myanmar, during 2007–2016 (Bai et al., 2018). From each patient, 2–3 ml of venous blood were drawn into a sodium citrate anticoagulant tube. The samples were transported to the laboratory at 4 °C for culture adaptation.

2.2. Parasite culture and in vitro drug assay

Parasite isolates adapted to long-term culture were subjected to in vitro sensitivity assays. LMF was purchased from Shanghai Macklin Biochemical Co., Ltd (Shanghai, China). A stock solution of 800 nM LMF was prepared in ethanol. Parasite cultures were maintained in O-type human red blood cells (RBCs) in a complete medium under an atmosphere of 92% N2, 3% O2, and 5% CO2. Cultures of synchronized ring-stage parasites were diluted with fresh complete medium to 2% hematocrit and 0.5% parasitemia. SYBR Green I-based assay was used to determine the in vitro susceptibility of each parasite isolate to LMF (Smilkstein et al., 2004). Each parasite isolate was measured in three technical and two biological replicates, with the 3D7 strain included as an internal reference in all assays.

2.3. DNA extraction and sequencing of the target region of the Pfnfs1 gene

DNA was extracted from cultured parasites using QIAmp 96 DNA kit (QIAGEN, Valencia, CA, USA) following instructions from the manufacturer. We genotyped the parasites at the merozoite surface protein 1 (msp1) and msp2 genes to determine if the isolates were monoclonal infections (Yuan et al., 2013). The target region of the Pfnfs1 gene was amplified by conventional PCR using the forward primer 5’ - CAAAAAGAACGCACTCGATAA-3′ and reverse primer 5′- TTCCATAGTGGTCCTTCATC-3’. PCR reaction was performed in 25 μl containing 10 μl of 2 × Phanta Max Buffer, 0.5 μl of each dNTP, 0.5 μl of each primer, 0.5 U of Super-Fidelity DNA polymerase P505d (Nanjing Vazyme Biotech, China), and 1 μl (5–10 ng) of DNA template. PCR cycling parameters were as follows: initial denaturation 5 min at 94 °C, 28 cycles at 94 °C for 30 s, 50 °C for 30 s, 68 °C for 40 s, and then a final extension at 68 °C for 7 min. The PCR products were sequenced using the Sanger sequencing method by Sangon Biotech Co. Ltd. (Kunming, China). We used the BioEdit software 7.0 to align sequence with the 3D7 reference sequence.

2.4. Statistical analyses

Statistical analysis was performed using GraphPad Prism 6.0 for Windows. The geometric mean of the half-maximal inhibitory concentration (IC50) was calculated by curve fitting. The Mann-Whitney U test was used to compare the two regions. The Chi-square test was used to compare the prevalence of genetic variation between the two regions.

3. Results

3.1. In vitro drug susceptibility to LMF

We collected 82 P. falciparum clinical samples from malaria patients with a recent travel history to Ghana and successfully cultured 29 isolates. In addition, 88 P. falciparum clinical samples from the China-Myanmar border were successfully cultured. The cultured isolates were assayed for in vitro susceptibilities to LMF. Overall, there was no significant difference between the field parasite isolates and 3D7 for IC50 values of LMF (p > 0.05, Mann-Whitney U test) (Table 1). However, isolates from the Ghana and the China-Myanmar border area differed significantly in susceptibilities to LMF (p = 0.0202, Mann-Whitney U test) (Table 1, Fig. 1). It is noteworthy that the IC50 values of individual parasite isolates varied widely; the IC50 values between the least and most susceptible isolates differed by 74.5-fold (Table 1).

Table 1.

In vitro susceptibilities (IC50 in nM) of culture-adapted field isolates from Ghana and China-Myanmar border to lumefantrine (LMF).

Drug Ghana (N = 29)
 China-Myanmar border (N = 88)
 P-valuea Total (N = 117)
 3D7
 P-valuea
Median (IQR) Range Median (IQR) Range Median (IQR) Range Mean ± SD
LMF 2.6 (2.0–3.3) 0.2–4.6 3.6 (2.2–5.7) 0.5–14.9 0.0202 3.0 (2.1–4.7) 0.2–14.9 2.2 ± 0.3 0.1000
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IQR –interquartile range.

SD – standard deviation.

a

Comparison between the two sites and total field isolates with 3D7 was done by the Mann-Whitney U test.

Fig. 1.

Fig. 1

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Dot plots showing in vitro susceptibilities of parasite isolates from Ghana and the China-Myanmar border area to lumefantrine (LMF). Each point represents the result for a single isolate. Median IC50 values and inter-quartile range are shown by the horizontal bars. * indicates P < 0.05.

3.2. Analysis of the Pfnfs1 gene

A 345 bp fragment of the Pfnfs1 gene was PCR amplified and sequenced from 117 isolates, of which 13.7% had reference K65 allele. For the 29 parasites collected from Ghana, 34.5% carried the reference K65, which was significantly higher than the frequency of the K65 allele from the China-Myanmar border samples (p = 0.0002) (Table 2).

Table 2.

Prevalence of the Pfnsf1 gene K65Q mutation in Ghana and China-Myanmar border.

Genes Mutation Total (n = 117) Ghana (N = 29) China-Myanmar border (N = 88) P-valuea
Pfnsf1 K65Q 86.3 65.5 93.2 0.0002
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a

χ2 test.

3.3. Association of the K65 allele with in vitro susceptibility of LMF

We compared the LMF IC50 values between parasites from each region carrying the Pfnfs1 reference K65 and non-reference 65Q alleles but observed no significant difference (p > 0.05) (Fig. 2).

Fig. 2.

Fig. 2

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Lack of association of the Pfnsf1 K65Q mutation with in vitro susceptibilities to lumefantrine (LMF). ns indicates not significant (P > 0.05, Mann Whitney U test).

4. Discussion

The use of molecular markers is crucial for drug resistance monitoring so that malaria control agencies can make timely policy adjustments. In this study, we tested in vitro susceptibilities of malaria parasites collected from West Africa and Southeast Asia to the ACT partner drug LMF. We found that the LMF IC50 values of the clinical isolates from the China-Myanmar border were significantly higher than those of the Ghanaian parasites. This difference may be related to the divergent antimalarial drug histories of the two regions. LMF is an antimalarial drug produced in China and was used in the China-Myanmar border area as early as the 1990s (Che et al., 1994a, 1994b). While AL was recommended as a first-line antimalarial drug by Myanmar in 2002 (WHO, 2008), the most frequently used drug in the China-Myanmar border area is DHA-PPQ. AL was recommended as a first-line drug in Ghana in 2008 (Abuaku et al., 2012), and it remains highly efficacious (90%) in treating uncomplicated falciparum malaria in Ghana. Although AL is still efficient in Myanmar (Han et al., 2020), day-3 parasite positivity was detected in more than 10% of patients after AL treatment in western Myanmar (Wu et al., 2020). This may reflect the increasing prevalence of artemisinin partial resistance in the GMS.

The Pfnfs1 gene has been associated with in vitro LMF resistance in parasites from The Gambia, with the reference K65 allele conferring higher IC50 values (Amambua-Ngwa et al., 2018). Further, the reference allele of the Pfnfs1 gene has been increasing in The Gambia, which was speculated to be due to strong selection pressure from the widespread use of AL (Amambua-Ngwa et al., 2018). We found that the Ghanaian parasites (34.5%), also from West Africa, had a much higher prevalence of the reference allele than the Myanmar parasites (6.8%), which is consistent with the more heavily deployed AL in Ghana. However, our analysis of the samples derived from two geographic regions did not find such an association, suggesting that the K65Q mutation may not be the best predictive marker for LMF resistance in P. falciparum. Yet, this study cannot rule out the potential role of Pfnfs1 gene polymorphism in LMF resistance for the following reasons. Firstly, the K65Q mutation in the Pfnfs1 gene may have a minor effect on LMF resistance, depending on the genetic backgrounds of the parasites. Secondly, this mutation may affect the fitness of the parasites, as shown for the pfaat1 mutations on CQ resistance (Amambua-Ngwa et al., 2023). Thirdly, LMF resistance may be a multigenic trait. In this case, additional mutations conferring LMF resistance would only be present in West Africa (Amambua-Ngwa et al., 2018) but not in Southeast Asia. Our research has some limitations. For travelers importing malaria, we do not know the locality at which they received an infective mosquito bite; thus, the parasite population studied was ill-defined. Due to the relatively small number of patients returning from West Africa, it may not be robust enough to establish the association between the Pfnfs1 mutation and the LMF susceptibility. Therefore, genetic manipulation in different genetic backgrounds and further study with a larger sample size are needed to clarify the role of the Pfnfs1 K65Q mutation in LMF resistance.

5. Conclusions

Thus, the potential effect of K65Q on LMF susceptibility needs to be evaluated by genetic manipulation in different genetic backgrounds, as has been done for the PfK13 mutations. As the Ghanaian samples used in this study may not be representative, continuous monitoring of the efficacy of antimalarial drugs and the prevalence of the Pfnfs1 mutation in Africa is warranted.

Ethical approval

All patients in this study voluntarily signed informed consent forms, and this study followed ethical principles according to the Helsinki Declaration. The research project was approved by the Institutional Review Committee of Kunming Medical University.

Funding

This study was supported by the National Science Foundation of China (31860604, U1802286 and 32370543), a grant (U19AI089672) from the National Institutes of Health, USA, Major science and technology projects of Yunnan Province (2018ZF0081), and International Science and Technology Cooperation-Yunnan International Science and Technology Cooperation Base (202003AE140004). XC, ZX, YZ and WZ were under sponsoring by the Yunnan Applied Basic Research Projects-UnionFoundation (202301AY070001-107, 2019FE001-015, 202101AY070001-108 and 202301AY070001-116 respectively). WH was supported by Guangxi Zhuang Autonomous Region Health Commission of Scientific Research Project (ZA20231282). WZ and XC was supported by the Education Department Fund of Yunnan Province (2019J1184 and 2023Y0606 respectively).

Availability of data and materials

All data generated or analyzed during this study are included in this published article. The clinical malaria isolates collected and cultured during the present study are available from the corresponding author on reasonable request.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Contributor Information

Liwang Cui, Email: liwangcui@usf.edu.

Zhaoqing Yang, Email: yangzhaoqing1@kmmu.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

All data generated or analyzed during this study are included in this published article. The clinical malaria isolates collected and cultured during the present study are available from the corresponding author on reasonable request.

Articles from International Journal for Parasitology: Drugs and Drug Resistance are provided here courtesy of Elsevier

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