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Molecular Epidemiology of Malaria in Cameroon. XXVIII. In vitro Activity of Dihydroartemisinin against Clinical Isolates of Plasmodium falciparum and Sequence Analysis of the P. falciparum ATPase 6 Gene

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  • 1 Unité de Recherche Maladies Infectieuses et Tropicales Emergentes, Institut de Recherche pour le Développement and Laboratoire de Recherche sur le Paludisme, Organisation de Coordination pour la Lutte Contre les Endémies en Afrique Centrale, Yaoundé, Cameroon; Antimalarial Drug Resistance, Global Malaria Programme, World Health Organization, Geneva, Switzerland

The Plasmodium falciparum ATPase 6 (Pfatp6), homolog of sarco-endoplasmic reticulum, calcium-dependent ATPase in malaria parasites, has been proposed to be the main target of artemisinins. Four distinct point mutations (L263E, E431K, A623E, and S769N) have been reported to be associated with artemisinin resistance. The Pfatp6 sequence polymorphism was determined to evaluate the prevalence of these mutations in fresh clinical isolates in Yaounde, Cameroon, and compare sequence data with in vitro response to dihydroartemisinin. Two major haplotypes were observed: the wild-type LEAS (n = 60, 62%) and a single mutant LKAS (n = 35, 36%). These amino acid substitutions did not influence the level of in vitro response to dihydroartemisinin (P > 0.05). Plasmodium falciparum isolates from Cameroon are highly sensitive in vitro to artemisinins. However, the relatively high prevalence of E431K may be a warning signal that warrants a regular monitoring of these molecular markers and/or in vitro activity of artemisinin derivatives.

INTRODUCTION

Artemisinins remain the main hope for current malaria control efforts because of their effectiveness for the treatment of multidrug-resistant Plasmodium falciparum infections and rapid action to treat severe and complicated malaria.1 As recommended by the World Health Organization,2 many countries where malaria is endemic, including those in Africa, have recently adopted, or are in the process of adopting, artemisinin-based combination therapy (ACTs) as the first-line drug for the treatment of uncomplicated malaria. This strategy improves antimalarial treatment effectiveness and is expected to delay the emergence of artemisinin-resistant parasites by rendering the probability of selecting drug-resistant parasites unlikely or very low. This assumption is based on the mutually protective action of artemisinin derivatives and other old and new antimalarial drugs, such as amodiaquine, sulfadoxine-pyrimethamine, mefloquine, lumefantrine, piperaquine, and pyronaridine.3 Artemisinins rapidly decrease the parasite biomass in the human host, and their drug partner, with relatively slower action, eliminates residual parasites.

The endoperoxide bridge has been demonstrated to be the key structure necessary for the schizontocidal action of artemisinins; its derivative lacking this bridge is inactive.4 The precise mechanism of action of artemisinins has not been elucidated. It has been suggested that a heme-dependent cleavage of endoperoxide bridge by iron-sulfur oxido-reduction within the food vacuole of the parasite and production of free radicals lead to alkylation and inhibition of functional parasite proteins.5 Another hypothesis is based on the classic enzyme-antagonist model in which artemisinins are thought to specifically inhibit a calcium-dependent ATPase, the sarco-endoplasmic reticulum calcium-dependent ATPase (SERCA) ortholog of P. falciparum ATPase 6 (Pfatp6), outside the food vacuole.6

Until recently, there have been only two documented cases of clinical resistance to artemisinins.7 Clinical failures to artemisinin monotherapies observed in the past have usually been ascribed to pharmacokinetic factors, in particular rapid elimination half-life and inadequate dosage.8 However, laboratory studies have shown that P. falciparum can develop stable resistance to artemisinin with high inhibitory concentrations (50% inhibitory concentration [IC50]), which suggest a potential for the emergence of in vivo drug resistance after a prolonged and extensive use of these drugs.9 Two candidate proteins that may explain artemisinin resistance have emerged in recent years. Several studies have suggested the possible role of gene amplification of or specific mutations in the P. falciparum multidrug-resistant 1 gene (pfmdr1) in artemisinin resistance (also mefloquine, halofantrine, and lumefantrine resistance). 1012 Other studies have not confirmed the association between pfmdr1 and artemisinin (and amino alcohol) resistance. 1316 An alternative basis of artemisinin resistance involving point mutation(s) in pfatp6 has been recently suggested. Using Xenopus laevis oocyte expression system and enzymology methods, Uhlemann and others found that L263E substitution can overcome SERCA inhibition by artemisinins. 17

Further in vitro studies that compared the low level of artemisinin sensitivity (artemether IC50 > 30 nM) in field isolates and pfatp6 sequences have suggested three amino acid substitutions that may be associated with artemisinin resistance: E431K and A623E in West Africa and S769N in South America. 18 It remains to be established whether these mutations lead to clinical resistance to artemisinins and/or ACTs. The aim of this study was to analyze Pfatp6 sequence polymorphisms in fresh clinical isolates collected from 2001 through 2006 in Yaoundé, Cameroon, and compare the data with dihydroartemisinin IC50s.

PATIENTS AND METHODS

Blood collection.

After informed consent was obtained, fresh clinical isolates of P. falciparum were collected by venipuncture (5–10 mL of blood in EDTA-coated collection tubes) from symptomatic patients ≥ 12 years of age who came to the Nlongkak Catholic missionary dispensary for febrile episodes in Yaoundé in 2001, 2002, 2003, and 2006. The inclusion criteria were a parasitemia ≥ 0.1%, absence of other Plasmodium species, and denial of recent self-medication with an antimalarial drug confirmed by the Saker-Solomons urine test. 19 Children < 12 years of age, pregnant women, and patients with signs and symptoms of severe and complicated malaria were excluded. The enrolled patients were treated with either amodiaquine monotherapy or an amodiaquine–sulfadoxine-pyrimethamine combination in 2002–2003 and artesunate–amodiaquine in 2006. The study was reviewed and approved by the Cameroonian National Ethics Committee and Cameroonian Ministry of Public Health.

In vitro assay.

Artemisinin was obtained from the Sigma Chemical Co. (St. Louis, MO). Several batches of dihydroartemisinin were obtained from Sapec (Lugano, Switzerland) and Shin Poong Pharmaceutical Co. (Seoul, South Korea) through the courtesy of the World Health Organization (Geneva, Switzerland). Stock and working solutions were prepared in methanol and distributed in triplicate in 96-well culture plates and dried before use, as described in previous work. 20

The in vitro response was determined by the isotopic microtest. 21 Blood samples were transported to our laboratory within 1–2 hours after collection. Infected erythrocytes were washed three times with RPMI 1640 medium buffered with 25 mM N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES) and 25 mM NaHCO3 by centrifugation and suspended in the complete RPMI 1640 medium with 10% non-immune, type AB + human serum (batch pooled from donors at the Blood Transfusion Center, Strasbourg, France for assays performed before 2004; batch no. S02909S4190; Bio West Nuaillé, France, for assays after 2004) at a 1.5% hematocrit. 3H-hypoxanthine (1 μCi/well; Amersham International, Little Chalfont, United Kingdom) was added to the blood-serum-medium mixture to measure parasite growth. The suspension (200 μL) was distributed into each well. Parasitemia was adjusted to 0.6% by adding fresh uninfected erythrocytes if the initial parasitemia was ≥ 1%. The culture plates were incubated at 37°C in either a 5% CO2 incubator (before 2004) or candle jar (after 2004) for 42 hours. The plates were frozen to terminate the in vitro assay. Incorporation of 3H-hypoxanthine was measured with a liquid scintillation counter (Wallac 1409; Pharmacia, Uppsala, Sweden). The IC50, defined as the drug concentration at which 50% of the incorporation of 3 H-hypoxanthine is inhibited compared with that of drug-free control wells, was calculated by a nonlinear regression analysis using Prism™ software (GraphPad Software, Inc., San Diego, CA).

Polymerase chain reaction and DNA sequencing.

After performing in vitro assays, remaining erythrocyte pellet was frozen at −20°C in aliquots of 1–2 mL and stored. Blood samples were thawed to extract parasite (and human) DNA. Infected erythrocytes were suspended in 15 mL of ice-cold lysis buffer (150 mM NaCl, 10 mM EDTA, 50 mM Tris, pH 7.5 [NET buffer], 0.015% saponin). The lysate was centrifuged at 2,000 × g for 10 minutes, and the pellet was transferred to a 1.5-mL microfuge tube and suspended in 500 μL of NET buffer. The mixture was incubated with 1% N-lauroylsarcosine and RNAse A (100 μg/mL) at 37°C for 1 hour, followed by an incubation with proteinase K (200 μg/mL) at 50°C for 1 hour. DNA was extracted three times in equilibrated phenol, pH 8, phenol-chloroform-isoamyl alcohol (v/v/v 25:24:1), and chloroform-isoamyl alcohol (v/v 24:1) and precipitated by the addition of 0.3 M sodium acetate and cold absolute ethanol. The extracted DNA was air-dried and resuspended in 50–100 μL of TE buffer (10 mM Tris, 1 mM EDTA).

The complete DNA sequence of the Pfatp6 gene is 4,049-basepairs (bp), and it is located in chromosome 1. The gene contains three exons and three introns. A 1,793-bp fragment spanning the coding region of exon I of the Pfatp6 gene was amplified by polymerase chain reaction (PCR). Specific synthetic oligonucleotides were designed from the DNA sequence of the K1 P. falciparum strain (GenBank accession no. AB121051). The reaction mixture for the primary PCR consisted of 3 μL of DNA, 15 picomol of the forward primer Pfatp6-1F, 5′-TTTATTTTCATCTACCGCTATTGTATGTGG-3′ and reverse primer Pfatp6-2R, 5′-GCATTTATACATCCTTCT CGTTAATCTAAT-3′, 200 mM of deoxynucleoside triphosphate mixture (dGTP, dATP, dTTP, and dCTP), 2.5 mM MgCl2, 50 mM KCl, 10 mM Tris buffer, pH 8.4, and 1 unit of Taq DNA polymerase (Roche Diagnostics, Meylan, France) in a final volume of 50 μL. Amplification was performed using a PTC-100 thermal cycler (MJ Research, Watertown, MA) under the following conditions: denaturation at 94°C for 2 minutes for the first cycle and 1 minute in subsequent cycles, annealing at 50°C for 2 minutes for the first cycle and 1 minute in subsequent cycles, and extension at 72°C for 3 minutes for all cycles, for a total of 35 cycles.

The secondary nested PCR was performed with a similar PCR mixture as for the primary amplification using 1 μL of the primary amplification product and 30 picomol of the first internal primer pair Pfatp6-5F (5′-GACTGAAATAGGTCATATTCAGCAT GC-3′) and Pfatp6-6R (5′-CATTTTTATTTTCTTACTATCAC TAG-3′) or the second primer pair Pfatp6-10F (5′-CACCTGTA CAATCATCAAATAAGAAGG-3′) and Pfatp6-12R (5′-CTT CTAATTTATAATAATCATCTGTATTC-3′) in a final volume of 100 μL. The primer pair Pfatp6-5F/Pfatp6-6R yields a 652-bp fragment spanning codons 263 and 431, which were reported to be associated with artemisinin resistance. The primer pair Pfatp6-10F/Pfatp6-12R amplifies a 645-bp fragment spanning codons 623 and 769. The following thermal cycling program was used for secondary amplification: denaturation at 94°C for 2 minutes for the first cycle and 1 minute for subsequent cycles, annealing at 50°C for 1 minute for all cycles, and extension at 72°C for 1 minute for all cycles, for a total of 35 cycles. The amplified nested PCR products were visualized by agarose gel electrophoresis and ultraviolet transillumination. Each secondary amplification product was sequenced from the 5′- and 3′-ends by using the automated DNA sequencer (ABI Prism; Perkin Elmer Corp., Les Ulis, France).

Data analysis.

The in vitro sensitivity to artemisinin derivatives was expressed as the geometric mean IC50. The threshold value for artemisinin resistance in vitro is not established. The mean IC50s during three study periods (2001–2002, 2003, and 2006) were compared by the one-way analysis of variance. The geometric mean IC50s for dihydroartemisinin were compared between groups of isolates with different Pfatp6 haplotypes using the Student t-test. The significance level of all statistical tests was set at 0.05.

The deduced amino acid sequences of the isolates from Cameroon were compared with those of P. falciparum reference clones 3D7 (GenBank accession no. PFA0310c) and K1 (GeneBank accession no. AB121051). Sequence alignment was performed using MacDNASIS Pro software (Hitachi Software Engineering Co., Yokohama, Japan). The following amino acid substitutions were analyzed for each isolate: L263E, E431K, A623E, and S769N. The presence of novel amino acid residues was also noted. The amino acid substitution observed in some Thai isolates (I89T) was not studied because it was shown to be unrelated to drug sensitivity. 12

RESULTS

The in vitro responses to dihydroartemisinin were available for 234 isolates from 2001 through 2006 (n = 90 in 2001–2002, n = 93 in 2003, and n = 51 in 2006). In vitro data for 2001–2003 have been reported in previous work. 22 The geometric mean IC50s (95% confidence interval [CI], [range]) were 1.29 nM (1.09–1.53 nM [0.220–5.88 nM]) in 2001–2002, 0.585 nM (0.475–0.721 nM [0.074–8.21 nM]) in 2003, and 3.30 nM (2.66–4.07 nM [0.636–18.4 nM]) in 2006. The significantly higher dihydroartemisinin IC50s in 2006 (P < 0.05) were caused by the change in the batch of pooled human serum and, to a lesser extent, incubation in a candle jar, rather than in a 5% CO2 incubator that had been used before 2004 in our laboratory. In 2006, in vitro response to artemisinin was also determined in parallel with that of dihydroartemisinin. The mean geometric mean IC50 (95% CI [range]) for artemisinin was 5.64 nM (4.61–8.51 nM [1.71–38.1 nM]) in 2006.

The Pfatp6 sequences of 97 isolates collected in 2002, 2003, and 2006 were determined (Tables 1 and 2). Based on codons (263, 431, 623, and 769) that have been associated with artemisinin resistance in previous studies, 17,18 two major haplotypes have been observed in this study: the wild-type LEAS (n = 60) and a single mutant LKAS (n = 35). An additional amino acid residue (E432K) that has not been previously reported was observed in two isolates. The haplotypes (263, 431, 432, 623, and 769) of these isolates were the single mutant LEKAS and the double mutant LKKAS. The West African-type double mutant (E431K + A623E) or the South American-type single mutation (S769N) was not found among isolates from Cameroon. With the exception of these five codons, sequence analysis showed a highly conserved nucleotide sequence, identical to 3D7 sequence, among all isolates within the regions spanning codons 259–454 and 588–790.

The comparison of geometric mean dihydroartemisinin IC50s of isolates carrying LEAS haplotype (1.55 nM) and those with LKAS haplotype (1.38 nM) showed no significant difference (P > 0.05), suggesting that the single amino acid substitution at this position is not associated with artemisinin resistance. Further comparison of dihydroartemisinin IC50s in the subsets of isolates by year (2002–2003 and 2006) showed no significant difference (P > 0.05) between the two major haplotypes found in this study. The presence of the novel mutation E432K with or without the E431K change resulted in IC50s (0.498 nM and 3.67 nM) within the range of IC50s of isolates with wild-type Pfatp6.

DISCUSSION

The present study suggests a high in vitro activity of artemisinin derivatives against clinical isolates of P. falciparum from Cameroon over the past 13 years (1994–2006). 2224 Dihydroartemisinin and artemether IC50s have consistently been < 20 nM at our study site. Other in vitro studies based on 3H-hypoxanthine radioisotope assay have shown the high activity of artemisinin derivatives in Africa. 2527 Few isolates > 30 nM, displayed increased IC50s for artemisinin derivatives even in Asian countries where these drugs have been used more extensively than in Africa. 18,2729

Several recent studies have analyzed Pfatp6 sequences in clinical isolates. In studies conducted on isolates from Tanzania, all codons at positions 263 (n = 355 isolates), 623 (n = 288) and 769 (n from pooled results = 552) were wild type. 30,31 Among P. falciparum isolates from Asia, none had mutations in codons 769 (n = 95 isolates from China and 14 isolates from Cambodia) and 263 (n = 14 isolates from Cambodia). 31,32 In a study based on imported malaria from various African countries, 33 no amino acid substitution was also found at position 263 among 154 isolates, whereas only one fully sensitive isolate (dihydroartemisinin IC50 = 0.83 nM) carried the South American-type S769N substitution. Our results are in agreement with these recent studies on the analysis of Pfatp6 codons 263 and 769 in P. falciparum isolates from Africa and Asia.

The West African-type E431K and A623E double substitutions were not analyzed in all previous studies. In the present study, E431K single substitution was found in 37% of isolates from Cameroon. Dahlström and others 31 reported a similar finding, with the presence of E431K (mixed alleles E431K + A623E in 2 isolates) in 31% of samples from Zanzibar and 39% from mainland Tanzania but these investigators did not provide in vitro response data to correlate their results. Our data suggest that E431K substitution, without A623E change, is insufficient to decrease in vitro sensitivity to dihydroartemisinin, as first observed by Jambou and others. 18 Other Pfatp6 mutations (both synonymous and non-synonymous) have been reported. This gene seems to be more polymorphic in East Africa (Tanzania), with at least 25 amino acid substitutions, mostly in a very limited number of isolates, and possibly in West Africa (Senegal; exact number and positions of mutations unreported), than in central Africa, but the relevance of these mutations to artemisinin resistance is unknown. 18,31

Most importantly, two molecular indicators of artemisinin resistance previously found in P. falciparum isolates, the South American-type Pfatp6 S769N and the West African-type Pfatp6 double mutation E431K + A623E, 18 were absent in isolates from Cameroon collected during 2002–2006. However, the presence of a single E431K mutation, found in approximately one-third of isolates from Cameroon and Tanzania, may be a warning signal that warrants a regular monitoring of these molecular markers and/or in vitro activity of artemisinin derivatives.

Interpretations of some recent experimental data imply that artemisinins may interact with target(s) other than Pfatp6/SERCA. In an experimental model using Toxoplasma gondii, artemisinin resistance was not associated with SERCA sequence changes or expression level but was associated with alterations in cytosolic calcium. 34,35 Moreover, a study has shown the same in vitro activities of enantiomeric pairs of trioxanes (synthetic artemisinin analogs) against P. falciparum, leading the investigators to conclude that the iron-dependent endoperoxide activation occurs without stereospecific interaction between the drug and its still unidentified target and that artemisinin does not interact with a specific protein target, including SERCA. 36 More laboratory evidence, including studies on PfATP6 structure and interaction with artemisinins, 37 is needed to confirm the pivotal role that Pfatp6 may play in artemisinin resistance.

The available in vitro and molecular data on artemisinin activity and Pfatp6 sequences are indirectly supported by the generally high efficacy of ACTs in Africa. Most clinical studies based on a 28-day follow-up and PCR adjustment and distinction between recrudescence and reinfection have reported only a few cases of treatment failures. 7,38 The causes of treatment failures are often ascribed to pharmacokinetic and/or pharmacodynamic variations. In this context, further studies are needed to correlate Pfatp6 sequence variations, in vitro artemisinin response, therapeutic efficacy of ACT (or if allowed, artemisinin monotherapy), and plasmatic drug concentrations. Moreover, as long as there is no documented evidence from the field that Pfatp6 mutations can fully explain artemisinin resistance, in vitro and in vivo, research on other candidate drug resistance genes should be pursued.

Table 1

In vitro dihydroartemisinin sensitivity of Plasmodium falciparum isolates with wild type Pfatp6 codons, Cameroon*

Table 1
Table 2

In vitro dihydroartemisinin sensitivity of Plasmodium falciparum isolates from Cameroon with mutant Pfatp6 codons*

Table 2

*

Address correspondence to Leonardo K. Basco, Unité de Recherche Maladies Infectieuses et Tropicales Emergentes, Institut de Recherche pour le Développement and Laboratoire de Recherche sur le Paludisme, Organisation de Coordination pour la Lutte Contre les Endémies en Afrique Centrale, BP 288, Yaoundé, Cameroon. E-mail: lkbasco@yahoo.fr

Authors’ addresses: Rachida Tahar and Leonardo Basco, Unité de Recherche Maladies Infectieuses et Tropicales Emergentes, Institut de Recherche pour le Développement and Laboratoire de Recherche sur le Paludisme, Organisation de Coordination pour la Lutte Contre les Endémies en Afrique Centrale, BP 288, Yaoundé, Cameroon, E-mails: rachida.tahar@ird.fr and lkbasco@yahoo.fr. Pascal Ringwald, Antimalarial Drug Resistance, Global Malaria Programme, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland, E-mail: ringwaldp@who.int.

Acknowledgments: We are grateful to Sisters Marie-Solange Oko and Pauline Ngono Mbia and their nursing and laboratory staff at the Catholic missionary Nlongkak dispensary in Yaounde for invaluable help in recruiting patients.

Financial support: This study was supported by the European Union (Redox Antimalarial Drug Discovery [READ-UP] project, contract no. 018602) and Technical Service Agreement, Global Malaria Programme, World Health Organization (Geneva, Switzerland).

Disclaimer: Pascal Ringwald is a staff member of the World Health Organization. This author alone is responsible for the views expressed in this publication, and they do not necessarily represent the decisions, policy, or views of the World Health Organization.

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