JAC Advance Access originally published online on November 30, 2005
Journal of Antimicrobial Chemotherapy 2006 57(1):110-115; doi:10.1093/jac/dki420
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Apparent absence of atovaquone/proguanil resistance in 477 Plasmodium falciparum isolates from untreated French travellers
1 Centre National de Référence pour la Chimiosensibilité du Paludisme, APHP, Hôpital Bichat-Claude Bernard, Paris, France; 2 Laboratoire de Biologie Animale et Parasitaire, EA 209, Université Paris Descartes, Paris, France; 3 Unité de Recherche en Biologie et Epidémiologie Parasitaires, Institut de Médecine Tropicale du Service de Santé des Armées, Parc du Pharo, Marseille, France; 4 Institut Fédératif de Recherche 48, Marseille, France; 5 Unité de Recherche en Pharmacogénétique Parasitaire, Institut Médecine Tropicale du Service de Santé des Armées, Parc du Pharo, Marseille, France
* Correspondence address. Laboratoire de Parasitologie, Hôpital Bichat-Claude Bernard, 46 Rue Henri Huchard, 75877 Paris cedex 18, France. Tel: +33-140-257-899; Fax: +33-140-256-763; E-mail: jacques.lebras{at}bch.ap-hop-paris.fr
Received 5 March 2005; returned 7 July 2005; revised 22 July 2005; accepted 24 October 2005
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Abstract |
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Objectives: We examined the atovaquone in vitro susceptibility and the cytochrome b (cytb) gene polymorphism of African Plasmodium falciparum isolates during the first years of atovaquone/proguanil use.
Patients and methods: Between 1999 and 2004, we collected blood samples from French P. falciparum-infected patients returning from African countries. Atovaquone susceptibility was determined using an in vitro isotopic test and cytb genotyping was performed by restriction fragment length polymorphism analysis and sequencing. These results were analysed according to the clinical response to atovaquone/proguanil treatment.
Results: No in vitro atovaquone resistance (IC50 > 1900 nM) and no cytb mutation leading to the Y268S substitution were detected among 477 unexposed African P. falciparum isolates. Eight cytb polymorphisms were found outside the ubiquinone reduction site by sequencing the entire gene of 270 isolates. One atovaquone/proguanil treatment failure was documented; the post-treatment isolate had an atovaquone susceptibility of 8230 nM and the Ser268 Cytb change; the pre-treatment isolate, obtained 4 weeks previously, was Cytb Tyr268 (wild-type).
Conclusions: No atovaquone/proguanil resistance was detected by phenotyping or genotyping among 477 unexposed African P. falciparum isolates. Atovaquone/proguanil-resistant parasite was detectable only in the post-treatment isolate from a treatment failure.
Keywords: susceptibility , malaria , antimalarials , P. falciparum
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Introduction |
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Two billion people live in malaria endemic countries. Around 400 million clinical episodes and two million deaths occur in Africa every year, mostly during childhood. Fifty million travellers from developed countries visit malaria endemic areas every year, leading to 30 000 imported cases of the disease, 12 000 of which are diagnosed in Europe.1 The emergence and spread of multidrug-resistant plasmodia calls for urgent development of new drugs with original cellular targets.2 Atovaquone is the first metabolically stable hydroxynaphthoquinone with a broad spectrum of activity against protozoan parasites, including plasmodia.3 In the mid-1990s preliminary clinical trials showed rapid defervescence and Plasmodium falciparum clearance, but early relapse occurred in 30% of cases.4,5 A partner drug was thus sought to bolster atovaquone activity. Tetracycline, pyrimethamine and proguanil were found to be synergistic, and the efficacy, tolerability and safety of the atovaquone/proguanil combination was subsequently demonstrated, with a cure rate of more than 98% in patients with non-severe malaria.6,7 Malarone, which consists of a fixed-dose of the atovaquone/proguanil combination, was registered in North America and Europe (1996) for the treatment and prophylaxis of malaria. Owing to its high cost, its use is currently limited to travellers from industrialized countries.
In plasmodial mitochondria, ubiquinone carries electrons from dihydroorotate dehydrogenase to the respiratory chain, via the cytochrome bc1 complex.8 Atovaquone, which mimics ubiquinone, inhibits electron transfer by binding cytochrome b (Cytb).9 In the absence of electron transfer, the inner mitochondrial membrane potential collapses and, without dihydroorotate dehydrogenase oxidization, pyrimidine biosynthesis is inhibited.10,11 Proguanil has been used against malaria since the 1950s.12 After metabolization of proguanil into cycloguanil, the latter inhibits plasmodial dihydrofolate reductase (DHFR).13 Surprisingly, combined with atovaquone, unmetabolized proguanil by itself lowers the effective concentration at which atovaquone collapses the mitochondrial membrane potential.14 However, the molecular basis of this enhancement is unclear. Since the introduction of atovaquone/proguanil, 11 treatment failures have been reported in travellers returning from Africa. Seven of the eleven cases were associated with a change in codon 268 of Cytb.15–21 Mutations in the dhfr gene (i.e. cycloguanil resistance) had no effect on proguanil activity.22 Conversely, mutations in the cytochrome b gene (cytb) induced resistance to atovaquone and its combination.23 On this basis, and in the absence of a well defined protein target for proguanil, atovaquone/proguanil resistance is assumed to be linked to atovaquone resistance.
As for tuberculosis and AIDS, combinations are used for malaria treatment in order to minimize resistance and to prolong the activity of the few remaining effective drugs. More than ever, epidemiological monitoring of resistance is essential. Genotyping and in vitro phenotyping are commonly used methods. They necessitate reliable molecular markers and accurate in vitro susceptibility thresholds, respectively. Mutations in the cytb gene, leading to changes in codon 268, represent a potential molecular marker of atovaquone/proguanil resistance. To date, no atovaquone concentration threshold has been defined for in vitro resistance phenotyping. In this study, conducted during the first years of atovaquone/proguanil use, we investigated the baseline prevalence of P. falciparum susceptibility to atovaquone by measuring the dispersion of atovaquone 50% inhibitory concentration values (IC50s) and screening for cytb polymorphisms. The results, together with clinical responses to atovaquone/proguanil treatment, allowed us to define an in vitro atovaquone resistance threshold.
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Materials and methods |
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P. falciparum clones
We used the atovaquone-resistant strain TM90C2b, presenting with an amino acid change in Cytb (Ser268) and isolated after atovaquone treatment failure, and the susceptible strains 3D7 and W2 (Tyr268).6 Each strain was further cloned by the limiting dilution method. Atovaquone in vitro susceptibility values were 8060 ± 45 nM, 1.52 ± 0.78 nM and 1.22 ± 0.88 nM for the clones of TM90C2b, 3D7 and W2, respectively.
Clinical P. falciparum isolates
Between 1999 and 2004, blood samples with parasitaemia >0.2% were collected in several hospitals in France, from patients with uncomplicated P. falciparum monoinfection returning from West Africa and the Indian Ocean. Patients having taken atovaquone/proguanil prophylaxis were excluded from the study on the basis of a standardized questionnaire. No informed consent was required for this study as all following procedures are part of the French national recommendations for care and surveillance of malaria.
Atovaquone pre-dosed plates
Atovaquone was obtained from GlaxoSmithKline (Evreux, France). Stock solutions were prepared in methanol. Two-fold serial dilutions in methanol were distributed in triplicate in flat-bottom 24-well plates, dried, stored in darkness at room temperature and used within 6 months. To confirm the batch of pre-dosed plates, atovaquone susceptibility was determined with strains 3D7 and W2. If an aberrant value was obtained, the batch was discarded.
In vitro assay
The in vitro atovaquone susceptibility level (IC50) was determined using the isotopic semi-microtest method.24 For each clone of P. falciparum, the susceptibility level was determined four times. For clinical isolates, simultaneous assays based on triplicate determinations were done. If the maximal atovaquone concentration (384 nM) was not sufficient to totally inhibit parasite growth, the test was repeated with higher concentrations (up to 12 800 nM); this time, atovaquone was added directly to the medium to avoid problems of solubilization. In all cases, atovaquone susceptibility was determined prior to treatment. In case of atovaquone/proguanil treatment failure, in vitro susceptibility to atovaquone was tested again when parasites reappeared in blood. More than 600 tests allowed us to determine the in vitro atovaquone susceptibility of 477 isolates. Conditions and duration of shipment of the blood samples are likely to explain the failures.
Determination of in vitro resistance threshold
When physicians considered monitoring of treatment possible, patients had clinical and biological examinations on days 3, 7 and 28, along with recommendations for seeking care in case of illness on the other days. Based on WHO criteria, adequate clinical and parasitological response (treatment success) was defined as disappearance of asexual parasites with fever within 3 days without reappearance before day 28.25 A recrudescence of parasitaemia and fever between day 4 and day 28 was defined as a late clinical failure. Results of the follow-up were available for 22 of the 58 patients treated with atovaquone/proguanil whose isolates were phenotyped (21 successes and 1 late clinical failure). IC50 values were interpreted according to these treatment responses, allowing definition of atovaquone in vitro threshold correlating with clinical response. The limit value of resistance was represented by the lower IC50 associated with a treatment failure. Prophylactic failure allegations were not used as failure criteria.
Artificial mixtures of clones
The capacity of PCR-sequencing and nested PCR-restriction fragment length polymorphism analysis (RFLP) to detect a minor resistant genotype (Y268S) were tested using artificial mixtures. Synchronous cultures of 3D7 and TM90C2b clones were diluted in fresh erythrocytes to obtain 1% parasitized cells. Mixed suspensions (50%, v/v) were then prepared at the following TM90C2b/3D7 ratios: 100:0, 99:1, 98:2, 95:5, 90:10, 75:25, 50:50, 25:75, 10:90, 5:95, 2:98, 1:99 and 0:100. Genotype ratios, after DNA extraction, were confirmed by the quantitative fragment analysis method.26 The PCR-sequencing and the nested PCR-RFLP methods detected Y268S representing more than 10 and 5% of the total parasite population in the mixture, respectively. As nested PCR-RFLP is more sensitive, this method was used to analyse the entire panel of isolates (n = 477). The entire cytb gene was examined by PCR-sequencing in the 270 isolates with the lowest atovaquone susceptibilities.
DNA extraction
Parasite DNA was extracted from 200 µL of blood by using the QIAamp DNA minikit, as recommended by the manufacturer (Qiagen, Hilden, Germany).
Amplification and sequencing of the P. falciparum cytb gene
To amplify the whole cytb gene, primers were designed from the complete P. falciparum mitochondrial DNA sequence (GenBank accession number M99416 [GenBank] ) using Oligo 4.0 software (Wojceich Rychlik, 1989). The reaction mixture contained 0.3 µM of each primer (sense, 5'-ATGAACTTTTACTCTATTAATT-3'; antisense, 5'-TTATATGTTTGCTTGGGAGCT-3'), 200 µM of each dNTP, buffer (50 mM KCl/10 mM Tris–HCl, pH 8.3/2 mM MgCl2) and 2.5 U of Thermus aquaticus DNA polymerase (AmpliTaq Gold, Perkin Elmer) plus 5 µL of DNA extract in a total volume of 50 µL. The samples were denatured for 5 min at 95°C prior to 40 cycles (95°C for 30 s, 55°C for 30 s and 72°C for 40 s). An ultimate primer extension was run for 5 min at 72°C. An amplicon of 1131 bp was purified with the Qiaquick PCR purification kit (PE Biosystems) and sequenced with the ABI PRISM Big Dye Terminator Cycle Sequencing Kit (Applera) according to the manufacturer's protocol. Fluorescent PCR products were sequenced in an ABI PRISM 3100 Genetic Analyzer (Applera).
Nested PCR-RFLP
This method was previously described.27
Clonal diversity analysis
When a treatment failure was observed, clonal composition of isolates before and after the failure was determined by analysing the polymorphism of the merozoite surface protein 2 gene (msp2). The highly polymorphic region of this gene was amplified using a fluorescent primer followed by analysis in an ABI PRISM® 310 Genetic Analyzer. Each clone was visualized as a peak, and characterized by the size of the msp2 PCR product. A quantitative estimation of the proportion of the clone in the parasite population was obtained from the area under the curve of the peak. This quantitative fragment analysis method allows detection of clones accounting for more than 2% of the whole.26
Data analysis
Geometric means were used to minimize the effects of outlier values. IC50 values were log-transformed to obtain a normal distribution. Statistical analysis was performed with Statview software (SAS Institute, Inc., Cary, NC, USA). Means were compared with Student's t-test or ANOVA, and variances with Fisher's or Levene's test.
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Results |
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Patients' ages ranged from 1 to 81 (mean 33 ± 16 years), and the male/female sex ratio was 2.9. Atovaquone susceptibility (IC50) ranged from 0.1 to 8230 nM, with a geometric mean of 1.79 nM and a median of 2 nM. All values determined before treatment were between 0.1 and 28 nM and were normally distributed after log-transformation (Figure 1). Values ranged from 0.15 to 6.8 nM in patients with atovaquone/proguanil treatment success, from 0.5 to 14.7 nM in patients lost to follow-up and from 0.1 to 28 nM in patients having received other treatments. One late atovaquone/proguanil treatment failure was observed on day 26 in a patient returning from Mali. Unfortunately, atovaquone susceptibility testing was unsuccessful before treatment in this patient. The post-treatment value of 8230 nM was more than two logs different from values in the other three groups.
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Most travellers were infected in French-speaking Central and West African countries (81%), whereas the remaining were infected in Madagascar (n = 19) or The Comoro Islands (n = 73). None of the patients reported having taken atovaquone previously. In the absence of atovaquone pressure in Africa, the natural dispersion of atovaquone IC50 values was studied for countries with sufficient data (Figure 2). The geometric means of atovaquone susceptibility were 1.9 ± 1.2, 1.7 ± 1.2, 1.7 ± 1.3, 1.8 ± 1.3 and 1.6 ± 1.3 nM for Cameroon, Ivory Coast, Mali, Senegal and The Comoro Islands, respectively, without atovaquone/proguanil in vitro resistance identified. No significant difference was observed between these countries.
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Including the case of atovaquone/proguanil failure, no mutation was detected by RFLP in the cytb gene of pre-treatment isolates (n = 477). One Y268S change was identified in the post-treatment isolate from the failure. To screen for polymorphisms in the entire cytb gene, 270 isolates were further sequenced. Nine single-nucleotide polymorphisms (SNPs) were identified (Table 1). Five of the nine base pair changes were non-synonymous, namely V54L (GTT to CTT), S70N (AGT to AAT), Y268S (TAT to TCT, previously found with RFLP), F306L (TTT to TTA) and H362Q (CAT to CAA) (GenBank accession numbers AY588279 [GenBank] , AY588280 [GenBank] , AY910012 [GenBank] , AY910013 [GenBank] and AY910014 [GenBank] ). The four other SNPs were synonymous. Eight SNPs occurred in isolates with atovaquone susceptibility values between 3.6 and 8.7 nM, the Y268S change was associated with an atovaquone IC50 of 8230 nM. msp2 profile of the pre-treatment isolate allowed identification of two clones accounting for 77% and 23% of the whole, respectively. After the failure, the initially dominant clone disappeared, the minor clone was still present at a low value (13%) and a new clone accounted for 87% of the whole.
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Discussion |
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We observed one late clinical failure during a 6 year atovaquone resistance phenotyping survey of P. falciparum. Unlike pre-treatment isolates, parasites recovered after failure bore a mutation modifying codon 268 of Cytb. This change was associated with an atovaquone IC50 of 8230 nM. Three previously published atovaquone treatment failures had in vitro susceptibilities of 1900 nM (after a standard atovaquone/proguanil regimen), 10 000 nM and 13 500 nM (after treatment with atovaquone alone or in various combinations).5,15,28 We also evaluated the atovaquone susceptibility of strain TM90C2b isolated from a Thai patient after atovaquone failure.6 These five values differed markedly from those associated with therapeutic successes (0.2–6.8 nM), creating a bimodal distribution distinguishing cure and failure. Consequently, all isolates studied before treatment may reasonably be considered as susceptible (0.1–28 nM).
We identified isolates with amino acid changes owing to SNPs. Except for Y268S, these changes were located outside the ubiquinone reduction site and were associated with high atovaquone susceptibility. This study being done during the first years of atovaquone/proguanil use, drug pressure was not involved in these SNPs, which probably represent natural polymorphisms. This low frequency of cytb polymorphism is consistent with the strong conservation of mitochondrial genes linked with functions crucial for parasite survival.29 In several Plasmodium species, Tyr268 is conserved at a position close to the ubiquinone reduction Q0 site. The change to serine, a hydrophilic amino acid, limits hydrophobic contacts with atovaquone. This could explain the consistent and marked decrease in atovaquone susceptibility in mutated parasites.15,28 As this Y268S change is not systematically detected after atovaquone/proguanil failure, poor atovaquone bioavailability is likely to explain most of these discrepancies.20,30 Nevertheless, in parasites from our patient with atovaquone/proguanil failure, two clones with distinct msp2 allelic profiles were identified before treatment. Genotyping results suggested that at least the majority clone, and probably the other one, were Tyr268. After treatment failure, one clone disappeared and a third one emerged. Considering its high proportion (87%), the corresponding genotype was probably linked to sequencing result, Ser268. As this Ser268 mutant and the two other clones differed in their msp2 gene, the mutant clone was probably present but undetectable before treatment. An alternative explanation is appearance of the mutation in the patient's parasites during treatment. This points to lesser fitness of Ser268 mutants in the natural situation, as previously observed with other cytb mutants in vitro.31 This also demonstrates the limits of genomic resistance detection, as minor clones may go undetected.26 We thus sequenced the cytb gene in residual P. falciparum parasites 1 day after the end of atovaquone/proguanil treatment. No changes were detected in comparison with the pre-treatment isolate (data not shown).
This work suggests that the atovaquone resistance threshold previously published (5–7 nM), before the emergence of atovaquone/proguanil resistance, should be reconsidered.32 This value was chosen after analysis of therapeutic responses, correlation with the quinine threshold, and use of the 90th percentile of atovaquone susceptibility. In our study, we observed a very high atovaquone IC50 (8230 nM) associated with the Y268S change in atovaquone/proguanil-resistant parasites. Considering our results and those of the literature, we recommend the use of the following two in vitro atovaquone thresholds to discriminate isolates from travellers: 0–30 nM, susceptible; >1900 nM, resistant. More cases of resistance are needed to adjust the cut-off, particularly with regard to the absence of intermediate values.
It has been suggested that antimalarial resistance within infected patients in a drug pressure environment develops in consecutive steps.33 In the first, the increase in resistance prevalence is not detectable, whereas the second step begins with clustered cases of resistance, followed by an exponential increase in resistance prevalence. A last phase may involve an increase in the level of resistance, as seen for resistance to DHFR or dihydropteroate synthase inhibitors, owing to an increase in mutation numbers. The usefulness of in vitro phenotyping and genotyping of pre-treatment samples is questionable during the first phase, particularly in view of our finding that resistant parasites are usually not detected before drug exposure. Cases of atovaquone/proguanil resistance being recently described in travellers, phenotyping and genotyping studies should be more informative during the therapeutic follow-up.
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Acknowledgements |
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Dennis Kyle (Walter Reed Army Institute of Research, Washington, DC, USA) and David Walliker (Cell Animal and Population Biology, Edinburg, UK) kindly provided us with strains TM90C2b and 3D7, respectively. We thank Philippe Deloron for helpful discussions and suggestions. This work was supported by the French Ministry of Health (grant to the National Reference Centre) and the French Armed Forces Health Service (grants DGA and DRT/STRDT). We thank GlaxoSmithKline for their support of in vitro atovaquone susceptibility measurements and blood drug assays in patients with Malarone® treatment failure through a 5 year Phase IV surveillance programme. L. M. is the recipient of a thesis fellowship from the French Research Ministry.
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References |
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