Open Access

No PfATPase6 S769N mutation found in Plasmodium falciparum isolates from China

Malaria Journal20087:122

https://doi.org/10.1186/1475-2875-7-122

Received: 22 April 2008

Accepted: 08 July 2008

Published: 08 July 2008

Abstract

Background

Artemisinin and its derivatives have been used for falciparum malaria treatment in China since late 1970s. Monotherapy and uncontrolled use of artemisinin drugs were common practices for a long period of time. In vitro tests showed that the susceptibility of Plasmodium falciparum to artemisinins was declining in China. A concern was raised about the resistance to artemisinins of falciparum malaria in the country. It has been reported that in vitro artemisinin resistance was associated with the S769N mutation in the PfATPase6 gene. The main purpose of this study was to investigate whether that mutation has occurred in field isolates from China.

Methods

Plasmodium falciparum field isolates were collected in 2006–2007 from Hainan and Yunnan provinces, China. A nested PCR-sequencing assay was developed to analyse the genotype of the PfATPase6 S769N polymorphism in the P. falciparum field isolates.

Results

The genotyping results of six samples could not be obtained due to failure of PCR amplification, but no S769N mutation was detected in any of the 95 samples successfully analysed.

Conclusion

The results indicate that the S769N mutation in the PfATPase6 gene is not present in China, suggesting that artemisinin resistance has not yet developed, but the situation needs to be watched very attentively.

Background

China has been severely afflicted by falciparum malaria. In 1954, falciparum malaria cases totaled about 4.18 million in the whole country. With active implementation of malaria control measures for more than 50 years, considerable success has been achieved [1]. However, up to the present, falciparum malaria is still an important public health problem in China, especially in Yunnan and Hainan provinces, with 3,240 cases reported from the two provinces in 2006, accounting for 93.4% of the total reported cases in the country [2].

The plant Artemisia annua (Asteraceae) has been used for more than 2,000 years in Chinese traditional medicine for the treatment of febrile illnesses, including malaria. Artemisia annua contains artemisinin, which was isolated in 1972, and since that time its efficacy against malaria has been amply demonstrated [3, 4]. Following increased chloroquine (CQ), piperaquine (PQ) and pyrimethamine-sulphadoxine (SP) resistance, artemisinin and its derivatives gradually became the mainstay of falciparum malaria therapy in China. Before the World Health Organization (WHO) urged pharmaceutical companies to end the marketing and sale of artemisinin for monotherapy in 2006 [5], many drug manufacturers in China distributed artemisinin alone to treat malaria; indiscriminate use of artemisinin derivatives was common practice. Therefore, whether resistance to artemisinins has developed in China is a cause for concern.

To this day, clinically relevant artemisinin resistance has not yet been documented, but it was found that the susceptibility of Plasmodium falciparum to artemisinin derivatives was declining in China, as IC50 for artesunate in 1999 were 3.3 times of that in 1988 [6]. Laboratory studies have shown that genetically stable and transmissible artemisinin-resistant rodent malaria parasite could be selected through prolonged exposure of drug-sensitive lines to low and increasing levels of artemisinins [7]. It is likely a matter of time before clinical artemisinin resistance is observed in China.

Recent studies provide compelling evidences that artemisinins act by selectively inhibiting PfATPase6 protein, the only SERCA-type Ca2+-ATPase in the P. falciparum genome, believed to be the primary target for artemisinins [8, 9]. A subsequent study in French Guyana showed that S769N PfATPase6 mutation was associated with raised artemether IC50, and suggested that the mutation may be used in molecular monitoring of artemisinin resistance to complement continuing in-vitro surveillance [10]. So far, no studies on this molecular marker have been carried out in China. The main purpose of the present study was to investigate whether there are any changes at codon S769N of the PfATPase6 locus in field samples from the country following the long-term use of artemisinins. A nested PCR-sequencing assay was developed to analyse the mutation of PfATPase6 S769N among P. falciparum field isolates collected from Hainan and Yunnan provinces, China.

Methods

Samples

Plasmodium falciparum field isolates were collected in 2006–2007 from two provinces. 27 samples were collected from Hainan province and 74 samples were from Yunnan province (Figure 1). The two provinces contributed more than 90% of the total falciparum malaria cases in the whole country in 2006 [2]. Parasite samples were collected from patients with uncomplicated P. falciparum infections before drug treatment. Diagnosis was carried out by microscopic examination of Giemsa-stained thick blood films. For each sample, approximately 20 μL of finger-prick blood was spotted on a piece of 3 MM filter paper (Whatman, Maidstone, UK) and air-dried. The dried filter paper samples were stored in individual zipper plastic bags with dryer at -20°C until DNA extraction. This study was reviewed and approved by the ethics committee of the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention.
Figure 1

Map of China showing the locations (shadow) where parasite samples were collected.

DNA extraction

Extraction of DNA from bloodspots on filter paper was carried out by the Chelex-100 (Bio-Rad Laboratories, Hercules, CA) boiling method described by Wooden and others [11] with some modifications described by Pearce and others [12]. The quality of DNA samples was tested by OD 260/280 measurement.

Nested PCR-sequencing assay

The fragment of pfATPase6 was amplified by nested PCR with primer F1 (5'-CAAATAAGAAGGATAAATCACCA-3') and primer R1(5'-TCATAAATACACGTATACCAGCC-3') (94°C for 3 min; then 94°C for 40 s, 46°C for 1 min, and 72°C for 1 min for 35 cycles; followed by a final extension step at 72°C for 10 min), and then primer F2 (5'-AAAATAAATACCACATCAACACAT-3') and primer R2 (5'-TCAATAATACCTAATCCACCTAAA-3') (94°C for 3 min; then 94°C for 40 s, 45°C for 1 min, and 72°C for 1 min for 35 cycles; followed by a final extension step at 72°C for 10 min). The 50 μL PCR mix contained primers at 0.25 μmol/L final concentration, 2 mmol/L MgCl2, 240 μmol/L of each deoxynucleoside triphosphate, and 2 U Taq polymerase. Template DNA (1 μL) was introduced to outer reaction mixtures. 1 μL outer PCR product was introduced into a 50 μL inner amplification mixture. Secondary PCR products were resolved by electrophoresis on 1% agarose gels and visualized by staining with ethidium bromide. Sequencing reactions were carried out using ABI PRISM Big Dye terminator v3.1 Cycle Sequencing kit (Applied Biosystems, CA, USA) as specified by the manufacturer's protocol. The sequences of the amplicons were aligned with the published data of the NCBI database by BLAST analysis.

Results

Nested PCR

A 437-base pair fragment in the PfATPase6 gene was amplified by nested PCR in field samples. Gel electrophoresis of the secondary PCR products indicated that they were of the expected sizes (Figure 2). Under this amplification conditions, 6 out of 101 samples could not be amplified.
Figure 2

Agarose gel electrophoresis of secondary PCR products. M, 100 bp DNA ladder. Lanes 1–5, P. falciparum samples; lane 6: Blank control.

Sequencing assay

Sequencing assays were successfully performed for the nested PCR products of 95 samples, no S769N mutation was detected in any of the analysed samples. Figure 3 shows a sequencing image of nested PCR product, indicating the polymorphism at codon 769 of the PfATPase6 gene.
Figure 3

Sequencing image of nested PCR product indicating the polymorphism at codon 769 of the PfATPase6 gene.

Discussion

In this study, by using nested PCR-sequencing assay, no PfATPase6 S769N mutation was found in any P. falciparum isolates collected from China. The finding is consistent with the results from the in vitro microtests [13, 14] and the high (more than 96%) cure rates of artemisinin combination therapies (ACTs) treatment for falciparum malaria in China [15, 16]. Accordingly, until now there is no sufficient evidence for assuming that artemisinin resistance has occurred in China. However, stable resistance to artemisinins has been induced in the laboratory, and the declining susceptibility of P. falciparum to artemisinin derivatives has been reported in the country [6]. Artemisinin drugs are believed as the most effective and the last hope in the near future in the battle against multidrug-resistant malaria, if resistance to artemisinins develops and spreads, it will be the most devastating event in the history of malaria control [17]. Therefore, it is wise to phase out artemisinin monotherapy in China; the use of ACTs recommended by WHO as first line treatment for uncomplicated malaria will either prevent, or at least delay, the development of artemisinin resistance.

Although evidences from laboratory and field studies have suggested that PfATPase6 protein is the primary target for artemisinins, and a specific S769N mutation in the gene was significantly associated with the reduced efficacy of artemisinins [810]. It should be pointed out that one laboratory study could not identify such mutation in artemisinin-resistant Plasmodium chabaudi [7]. Field studies carried out in Tanzania and Brazil also could not detect the S769N PfATPase6 mutation in the analysed samples [18, 19]. Thus, whether this mutation can be widely used as a reliable marker for artemisinin resistance in epidemiological studies needs further validation.

Conclusion

PfATPase6 S769N mutation was not detected in any of the P. falciparum isolates from China, no direct evidence supports that artemisinin resistance has occurred in the country until now. However, a high degree of vigilance is required; the level of artemisinin sensitivity of P. falciparum should be closely monitored.

Declarations

Acknowledgements

This work was supported by the Key Science-Technology Project of the National Tenth Five-year Plan of China (NO. 2004BA718B13). We are grateful to the on-site clinical and field officers, nurses and patients who consented to participate. We thank Prof. Yaoyu Feng for critically reading the manuscript.

Authors’ Affiliations

(1)
National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention

References

  1. Tang LH: Progress in malaria control in China. Chinese medical journal. 2000, 113 (1): 89-92.PubMedGoogle Scholar
  2. Zhou SS, Wang Y, Tang LH: [Malaria situation in the People' s Republic of China in 2006]. Chinese journal of parasitology & parasitic diseases. 2007, 25 (6): 439-411.Google Scholar
  3. Li GQ, Arnold K, Guo XB, Jian HX, Fu LC: Randomised comparative study of mefloquine, qinghaosu, and pyrimethamine-sulfadoxine in patients with falciparum malaria. Lancet. 1984, 2 (8416): 1360-1361. 10.1016/S0140-6736(84)92057-9.View ArticleGoogle Scholar
  4. Li GQ, Guo XB, Fu LC, Jian HX, Wang XH: Clinical trials of artemisinin and its derivatives in the treatment of malaria in China. Transactions of the Royal Society of Tropical Medicine and Hygiene. 1994, 88 Suppl 1: S5-6. 10.1016/0035-9203(94)90460-X.View ArticlePubMedGoogle Scholar
  5. Rehwagen C: WHO ultimatum on artemisinin monotherapy is showing results. BMJ (Clinical research ed. 2006, 332 (7551): 1176-10.1136/bmj.332.7551.1176-b.View ArticleGoogle Scholar
  6. Yang H, Liu D, Yang Y, Fan B, Yang P, Li X, Li C, Dong Y, Yang C: Changes in susceptibility of Plasmodium falciparum to artesunate in vitro in Yunnan Province, China. Transactions of the Royal Society of Tropical Medicine and Hygiene. 2003, 97 (2): 226-228. 10.1016/S0035-9203(03)90127-1.View ArticleGoogle Scholar
  7. Afonso A, Hunt P, Cheesman S, Alves AC, Cunha CV, do Rosario V, Cravo P: Malaria parasites can develop stable resistance to artemisinin but lack mutations in candidate genes atp6 (encoding the sarcoplasmic and endoplasmic reticulum Ca2+ ATPase), tctp, mdr1, and cg10. Antimicrobial agents and chemotherapy. 2006, 50 (2): 480-489. 10.1128/AAC.50.2.480-489.2006.PubMed CentralView ArticlePubMedGoogle Scholar
  8. Eckstein-Ludwig U, Webb RJ, Van Goethem ID, East JM, Lee AG, Kimura M, O'Neill PM, Bray PG, Ward SA, Krishna S: Artemisinins target the SERCA of Plasmodium falciparum. Nature. 2003, 424 (6951): 957-961. 10.1038/nature01813.View ArticleGoogle Scholar
  9. Uhlemann AC, Cameron A, Eckstein-Ludwig U, Fischbarg J, Iserovich P, Zuniga FA, East M, Lee A, Brady L, Haynes RK, Krishna S: A single amino acid residue can determine the sensitivity of SERCAs to artemisinins. Nature structural & molecular biology. 2005, 12 (7): 628-629. 10.1038/nsmb947.View ArticleGoogle Scholar
  10. Jambou R, Legrand E, Niang M, Khim N, Lim P, Volney B, Ekala MT, Bouchier C, Esterre P, Fandeur T, Mercereau-Puijalon O: Resistance of Plasmodium falciparum field isolates to in-vitro artemether and point mutations of the SERCA-type PfATPase6. Lancet. 2005, 366 (9501): 1960-1963. 10.1016/S0140-6736(05)67787-2.View ArticlePubMedGoogle Scholar
  11. Wooden J, Kyes S, Sibley CH: PCR and strain identification in Plasmodium falciparum. Parasitology today (Personal ed. 1993, 9 (8): 303-305. 10.1016/0169-4758(93)90131-X.View ArticleGoogle Scholar
  12. Pearce RJ, Drakeley C, Chandramohan D, Mosha F, Roper C: Molecular determination of point mutation haplotypes in the dihydrofolate reductase and dihydropteroate synthase of Plasmodium falciparum in three districts of northern Tanzania. Antimicrobial agents and chemotherapy. 2003, 47 (4): 1347-1354. 10.1128/AAC.47.4.1347-1354.2003.PubMed CentralView ArticleGoogle Scholar
  13. Liu L, Chen P, Ou F, Fu C: [In-vitro sensitivity of Plasmodium falciparum to the components of dihydroartemisinin compounds in dongfang city of hainan province]. Journal of Guangzhou University of Traditional Chinese Medicine. 2005, 22 (5): 377-379.Google Scholar
  14. Yang H, Liu D, Huang K, Dong Y, Yang Y, Yang P, Liao M, Zhang C, Liu R: [In vitro sensitivity of Plasmodium falciparum to derivatives of artemisinin, pyronaridine and chloroquine in Yunnan]. Chinese Journal of Parasitology and Parasitic Diseases. 1997, 15 (5): 292-296.Google Scholar
  15. Xu Y, Ou FZ, Chen PQ, Song JP, Fu LC: [Randomized trial of Artekin and Artekin(T) for treatment of uncomplicated falciparum malaria patients]. China Tropical Medicine. 2003, 3 (6): 723-725.Google Scholar
  16. Sun XD, Zhang ZX, Liu DQ, Huang GZ, Deng Y, Zhang CL, Wang SL, Li HB, Huang M, Jiang YC, Zhao LF, Xiao YH: [Clinical study on the efficacy of dihydroartemisinin and piperaquine in treatment of multi-drug resistant Plasmodium falciparum]. China tropical medicine. 2006, 6 (2): 211-212.Google Scholar
  17. Noedl H: Artemisinin resistance: how can we find it?. Trends in parasitology. 2005, 21 (9): 404-405. 10.1016/j.pt.2005.06.012.View ArticlePubMedGoogle Scholar
  18. Mugittu K, Genton B, Mshinda H, Beck HP: Molecular monitoring of Plasmodium falciparum resistance to artemisinin in Tanzania. Malaria journal. 2006, 5: 126-10.1186/1475-2875-5-126.PubMed CentralView ArticlePubMedGoogle Scholar
  19. Ferreira ID, Martinelli A, Rodrigues LA, do Carmo EL, do Rosario VE, Povoa MM, Cravo P: Plasmodium falciparum from Para state (Brazil) shows satisfactory in vitro response to artemisinin derivatives and absence of the S769N mutation in the SERCA-type PfATPase6. Trop Med Int Health. 2008, 13 (2): 199-207.View ArticleGoogle Scholar

Copyright

© Zhang et al; licensee BioMed Central Ltd. 2008

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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