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Screening for Mutations Related to Atovaquone/Proguanil Resistance in Treatment Failures and OtherImported Isolates of Plasmodium falciparumin Europe Ole Wichmann,1 Nikolai Muehlberger,1 Tomas Jelinek,1,3 Michael Alifrangis,14 Gabriele Peyerl-Hoffmann,1,3
Marion Mu¨hlen,1 Martin P. Grobusch,2,4 Joaquim Gascon,9 Alberto Matteelli,11 Hermann Laferl,19 Zeno Bisoffi,12
Stephan Ehrhardt,5 Juan Cuadros,10 Christoph Hatz,13 Ida Gjørup,15 Paul McWhinney,16 Jirˇi Beran,17
Saraiva da Cunha,18 Marco Schulze,6 Herwig Kollaritsch,20 Peter Kern,7 Graham Fry,21 and Joachim Richter,8
for the European Network on Surveillance of Imported Infectious Diseases
1Institute of Tropical Medicine, Charite´, Humboldt University, and 2Department of Medicine (Infectious Diseases), Charite´, Berlin, and 3Departmentof Infectious Diseases and Tropical Medicine, University of Munich, Munich, and 4Institut fu¨r Tropenmedizin, Eberhard-Karls-Universita¨t Tu¨bingen, Tu¨bingen,and 5Clinical Department, Bernhard Nocht Institute for Tropical Medicine, Hamburg, and 6Sta¨dtische Kliniken “St. Georg,” 2. Klinik fu¨r Innere Medizin,Leipzig, and 7Sektion Infektiologie und Klinische Immunologie, Universita¨t Ulm, Ulm, and 8Klinik fu¨r Gastroenterologie, Hepatologie, und Infektiologie,Medizinische Klinik und Poliklinik der Universita¨t, Du¨sseldorf, Germany; 9Seccio´n de Medicina Tropical, Hospital Clinic, Barcelona, and 10Departmentof Clinical Microbiology and Parasitology, Hospital Prı´ncipe de Asturias, Madrid, Spain; 11Clinica di Malattie Infettive e Tropicali, Universita´ di Brescia,Brescia, and 12Centro per le Malattie Tropicali, Ospedale S. Cuore, Negrar (Verona), Italy; 13Swiss Tropical Institute, Basel, Switzerland; 14Centre of MedicalParasitology, Panum Institute, and 15Department of Infectious Diseases, University Hospital, University of Copenhagen, Copenhagen, Denmark; 16BradfordRoyal Infirmary, Infection and Tropical Medicine, Bradford, United Kingdom; 17Department of Infectious Diseases, University Hospital, Hradec Kra´love´, CzechRepublic; 18Consulta de Medicina do Viajante, Departamento de Doenc¸as Infecciosas, Hospital Universita´rio, Coimbra, Portugal; 19Kaiser-Franz-Josef-Spitalder Stadt Wien, 4. Medizinische Abteilung mit Infektions und Tropenmedizin, and 20Abteilung fu¨r spezifische Prophylaxe und Tropenmedizin am Institutfu¨r Pathophysiologie, University of Vienna, Vienna, Austria; 21Tropical Medical Bureau, Dublin, Ireland Background.
Two single-point mutations of the Plasmodium falciparum cytochrome b gene (Tyr268Asn and Tyr268Ser) were recently reported in cases of atovaquone/proguanil (Malarone) treatment failure. However, littleis known about the prevalence of codon-268 mutations and their quantitative association with treatment failure.
Methods.
We set out to assess the prevalence of codon-268 mutations in P. falciparum isolates imported into Europe and to quantify their association with atovaquone/proguanil treatment failure. Isolates of P. falciparum collectedby the European Network on Imported Infectious Disease Surveillance between April 2000 and August 2003 wereanalyzed for codon-268 mutations, by use of polymerase chain reaction–restriction fragment–length polymorphism.
Results.
We successfully screened 504 samples for the presence of either Tyr268Ser or Tyr268Asn. One case of Ser268 and no cases of Asn268 were detected. Therefore, we can be 95% confident that the prevalence of Ser268 inthe European patient pool does not exceed 0.96% and that Asn268 is less frequent than 0.77%. In 58 patients treatedwith atovaquone/proguanil, Tyr268Ser was present in 1 of 5 patients with treatment failure but in 0 of 53 successfullytreated patients.
Conclusions.
Tyr268Ser seems to be a sufficient, but not a necessary, cause for atovaquone/proguanil treatment failure. The prevalence of both codon-268 mutations is currently unlikely to be 11% in the European patient pool.
Infected European travelers and immigrants carry a wide demic areas into the continent. Thus, if properly done, variety of Plasmodium falciparum strains from all en- data and parasite material from this population can beused to monitor the development of drug resistance inendemic areas, especially in sub-Saharan Africa [1].
A fixed combination of atovaquone and proguanil Received 4 March 2004; accepted 19 April 2004; electronically published 28 (Malarone; GlaxoSmithKline) is a drug that has been Reprints or correspondence: Dr. Ole Wichmann, Institute of Tropical Medicine, Spandauer Damm 130, 14050 Berlin, Germany (ole.wichmann@charite.de).
The Journal of Infectious Diseases
2004; 190:1541–6
ᮊ 2004 by the Infectious Diseases Society of America. All rights reserved.
Financial support: Friedrich Baur Stiftung, Ludwig-Maximilians-University (to P. falciparum Atovaquone/Proguanil Resistance • JID 2004:190 (1 November) • 1541
recently introduced for the treatment and prophylaxis of mul- ticular for correlation to the codon-268 mutations and in vivo tidrug-resistant P. falciparum malaria. Early evidence showed resistance. The cyt b gene mutations Tyr268Asn and Tyr268Ser that parasites may quickly develop resistance to atovaquone.
have been linked to cases of atovaquone/proguanil treatment One study showed that, when treated with atovaquone alone, failure [17–21]. To move molecular assays for point mutations 33% of patients had recrudescence of parasitemia [2]. It has on resistance-related genes into the realm of applied tools for been proposed that, because atovaquone inhibits electron trans- surveillance, we investigated a series of P. falciparum isolates port and collapses mitochondrial membrane potential at similar that had been imported into Europe between April 2000 and concentrations [3], this might lead to the formation of oxygen August 2003 for the prevalence of point mutations associated radicals, which could act as locally active mutagens [4].
with atovaquone/proguanil resistance.
When atovaquone is administered in combination with pro- guanil, cure rates of 99%–100% are achieved [5–10]. It has SUBJECTS, MATERIALS, AND METHODS
been shown that the biguanide itself, not the metabolic con- Sampling.
The study was established within the infrastructure version cycloguanil as an inhibitor of dihydrofolate reductase, of the European Network on Imported Infectious Disease Sur- synergizes with atovaquone by specifically lowering the con- veillance (TropNetEurop), which has been successfully provid- centrations at which atovaquone is able to collapse the mito- ing surveillance data on imported malaria since 1999 [22]. The chondrial membrane electropotential [11]. As a result, the in- network covers ∼12% of all imported cases of malaria in west- clusion of proguanil leads to an enhancement of atovaquone’s ern and central Europe. Sentinel surveillance reporting is cur- activity and reduces the chance of mutations arising in the rently done by 46 participating clinical sites throughout 16 European countries by use of a standardized and computerized There is evidence that atovaquone, on the basis of its struc- reporting system. Although the organization of the network tural similarity to ubiquinol, binds to the parasitic cytochrome does not guarantee a representative data collection for Europe, bc (cyt b) complex [12], and mutations in the cyt b gene of most referral centers in Europe are represented. A total of 18 the parasite mitochondrial genome have been described that centers sent in malaria isolates with their case notifications.
confer atovaquone resistance. Two mutations in Pneumocystis During standard malaria testing by thick and thin blood film, carinii at the ubichinol-binding pocket (the Q domain) have 10 mL of full blood was dotted on Whatman 3MM chroma- been shown to be associated with the failure of atovaquone tography paper and air-dried at room temperature before the prophylaxis [13]. Atovaquone-resistant Plasmodium yoelii lines initiation of treatment. DNA was prepared from the dried blood have been derived by subtherapeutic treatment of infected mice.
Five mutations near the putative atovaquone-binding pocket Polymerase chain reaction–restriction fragment–length poly-
have been identified at codons 258–272 [14]. In a similar study, morphism (PCR-RFLP) for codon-268 mutations.
3 mutations at the cyt b gene of atovaquone-resistant Plasmo- tection of resistance-related point mutations on the cyt b gene dium berghei lines were found to be associated with resistance was done according to protocols established elsewhere. For de- to atovaquone. In that study, mutations at codon 133 or 144, tection of the codon-268 mutations on cyt b, a PCR-RFLP in addition to an amino acid change at codon 284, led to in- method was used. Details have been published elsewhere [18].
A nested PCR was designed that used CYTb1 and CYTb2 as In studies with P. falciparum, atovaquone-resistant lines have outer primers and 3 different pairs of nested primers to dis- been derived in vitro by incubation at various concentrations tinguish the 3 known polymorphisms at codon 268. For the [16]. An initial mutation at codon 133 was found to confer a primary amplification reaction, a mix that contained 0.125 low resistance level that could be increased by additional mu- mmol/L each outer primer, 0.2 mmol/L dNTPs, 1.5 mmol/L tations in the codon 272–280 domain. In vivo, a P. falciparum Mg2+, and 0.5 U Taq polymerase (Qbiogene) was initially heat- isolate from a Thai patient with recrudescence after atovaquone ed at 94ЊC for 5 min and then cycled at 94ЊC for 50 s, 50ЊC and pyrimethamine treatment showed a mutation at codon 268 for 50 s, and 70ЊC for 1 min for 35 cycles, with a final extension (Tyr268Ser) of the cyt b gene [2, 16]. A different amino acid at 70ЊC for 5 min. For the secondary amplification, 1 mL of change at the same codon (Tyr268Asn) was described in an PCR product was added to the master mix that contained 0.5 English patient traveling to Nigeria who had failed atovaquone/ mmol/L primers and dNTP, MgCl , and Taq polymerase, as described above. PCR conditions were 94ЊC for 5 min, 30 cycles Protocols for the detection of relevant mutations have been of 95ЊC for 30 s, 55ЊC for 30 s, 72ЊC for 30 s, and a final developed and evaluated with in vitro isolates plus a few sam- extension at 72ЊC for 5 min for the primer pairs CYTb3/CYTb5 ples from patients with documented in vivo resistance. The and CYTb2/CYTb6. CYTb2/CYTb7 was annealed at 45ЊC. The results point convincingly toward correlations between the de- products of the second round were confirmed by electropho- tection of point mutations and phenotypic resistance, in par- resis in ethidium bromide–stained agarose gel.
1542 • JID 2004:190 (1 November) • Wichmann et al.
For RFLP analysis, 5 mL of PCR product was mixed with 1 Characteristics of the total study population.
U of the appropriate enzyme and its specific buffer in a total volume of 22 mL and incubated overnight at 37ЊC. The result was detected by electrophoresis in ethidium bromide–stained agarose gel. The primer pair CYTb3/CYTb5, when used in com- bination with the enzyme NsiI, cuts the wild type (wt) and the Asn268 mutation but not the Ser268 mutation. The primer pair CYTb2/CYTb6, used in combination with the enzyme AlwNI, cuts the Ser268 mutation but not the wt and the Asn268 mu- tation. The primer pair CYTb2/CYTb7, used in combination with the enzyme SspI, cuts the wt and the Ser268 mutation but The established P. falciparum laboratory clones K1 and FCR3, as well as our own in vitro isolates that are resistant to ato- vaquone/proguanil, were used as representative controls [18].
In cases where the PCR testing of samples did not reveal any result (neither wt nor mutation), the testing was repeated at least once. If the PCR result remained inconclusive, the testing was defined as unsuccessful, and the sample was excluded from Statistical analysis.
Results of the PCR testing of the P. falciparum isolates were individually matched with epidemio- logical and clinical data from the TropNetEurop surveillance da- Data are no. (%) of subjects, unless otherwise noted.
tabase and were analyzed by use of the statistical software SAS(release 8.01; SAS Institute). To be able to present statisticallyascertained estimates for mutation prevalence, even if no mu- marizes the characteristics of the 504 patients. The majority of tations were observed, maximum mutation prevalences were infections (54.2%) were acquired in West Africa, and most calculated that, given the study power, one could be 95% sure patients did not receive any malaria prophylaxis (82.0% of cases they were not surpassed. This may be interpreted as a 1-sided with available data). The ratio of non-Europeans (immigrants 95% confidence interval (CI). To derive the estimate, the num- or foreign visitors) to Europeans (either living in Europe or ex- ber of to-be-expected mutations that, under the assumption that the estimate was true, would make it !5% likely to find Characteristics of the treatment failures.
the observed number of mutations or fewer in the sample had mation was available for 329 of 504 patients. Of these, 253 re- to be determined. Under the assumption of Poisson distribu- ceived drugs other than atovaquone/proguanil, 18 received ato- tion of the mutation data, the probability could be derived vaquone/proguanil in combination with other drugs, and 58 from the distribution, which was defined as the number of received atovaquone/proguanil monotherapy. In the latter group, expected cases. Dividing the determined number of expected 5 treatment failures were reported. Of these, 3 cases of malaria cases by sample size yielded the maximum mutation prevalence.
had been acquired in West Africa, 1 in East Africa, and 1 inCentral Africa (table 2).
Patient A, a 30-year-old Gambian who is a resident of A total of 504 isolates of P. falciparum were screened for 2 Germany, was diagnosed with P. falciparum monoinfection (3% different mutations on codon 268 of the parasite’s cyt b gene, parasitemia) in September 2001 after returning from Gambia.
which had been previously associated with atovaquone/pro- He did not receive any malaria prophylaxis during the visit.
guanil treatment failure. Combining the results of the PCRs The patient was treated with atovaquone/proguanil for 3 days; with primer pairs CTYb3/CYTb5 and CYTb2/CYTb6, both of no parasites were detected in the thick blood film 8 days after which focus on the detection of the Ser268 mutation, a total the initiation of therapy. Three weeks after therapy, the patient of 495 samples were successfully screened for this specific mu- had a parasitological recrudescence without any symptoms.
tation. With the PCR using primer pair CYTb2/CYTb7, which Patient B, a 28-year-old male German, was diagnosed with focuses on the detection of the Asn268 mutation, 391 samples malaria in February 2002 in Mali, 8 days before he returned.
He received chloroquine and proguanil as malaria prophylaxis.
Characteristics of the total study population.
Treatment was provided with atovaquone/proguanil in ade- P. falciparum Atovaquone/Proguanil Resistance • JID 2004:190 (1 November) • 1543
Subsample of 58 travelers treated for malaria with
268 of all isolates. In 4 of 5 isolates, PCR results were additionally atovaquone/proguanil after returning to Europe.
confirmed by sequencing a 716-bp fragment of the cyt b gene[24], and no other variants were found.
Prevalence of codon-268 mutations.
tion, 1 case of Tyr268Ser (of 495) and 0 cases of Tyr268Asn (of 391) were detected. The prevalence of Tyr268Ser in patients treated in Europe for falciparum malaria between 2001 and 2003 can therefore be calculated with 95% confidence to be 0.01%– 1.12%. The maximum prevalence that will not be surpassed with 5% probability of error can be calculated as 0.96%. For Tyr- 268Asn, we can be 95% confident that the prevalence of this mutation does not exceed 0.77% in the European patient pool.
Association of codon-268 mutations with treatment failure.
In the subsample of 58 patients treated with atovaquone/pro- guanil, Tyr268Ser was present in 1 of 5 with treatment failure but in 0 of 53 successfully treated patients (table 2), which indicates that this mutation is not a necessary, but may be a sufficient, cause of atovaquone/proguanil treatment failure. Its presence was associated with a 14.3-times higher risk of atova- quone/proguanil treatment failure (relative risk [RR], 14.3 [95% Tyr268Asn was detected in none of the samples; however, in Data are no. (%) of subjects, unless otherwise noted.
6 of 58 samples, the testing for this specific mutation was not quate doses. Four weeks later, the patient developed a symp- successful, and the status therefore remained unknown. Ex- tomatic recrudescence with a density of 1.5% P. falciparum.
cluding these 6 samples from the analysis revealed a 12.8-times PCR-RFLP and sequencing revealed a mutation (Tyr268Ser) in higher risk for the occurrence of atovaquone/proguanil treat- ment failure in the presence of either of the 2 described codon- Patient C, a 33-year-old male German, was diagnosed with 268 mutations (RR, 12.75 [95% CI, 5.0–32.7]).
P. falciparum malaria after returning from a holiday in Kenyaand Tanzania in February 2003. He did not receive any malaria DISCUSSION
prophylaxis. The patient developed a febrile recrudescence 3 With growing international travel and the continued spread of weeks after directly observed treatment with atovaquone/pro- antimalarial drug resistance, the new fixed-dose combination of atovaquone and proguanil was most warranted as an agent that Patient D, a 56-year-old male Nigerian who is a resident is not only highly effective for the prophylaxis and treatment of of Spain, was diagnosed with P. falciparum malaria after re- multidrug-resistant P. falciparum malaria but is also well toler- turning from Nigeria in March 2003. The patient was treated ated, especially when it is used for prophylaxis [25, 26]. However, with atovaquone/proguanil and developed an early treatment early after its introduction, it was predicted that the combination failure with no negativity in the thick blood film.
of these 2 agents, which had already been in use for some time, Patient E, a 38-year-old Congolese woman who is a res- would be vulnerable to resistance in the near future [27]. This ident of Germany, was diagnosed with P. falciparum mono- is even more the case in drugs in which single- or double-point infection after returning from a trip to Kinshasa. She received mutations confer high levels of resistance.
only chloroquine for prophylaxis. Parasites recrudesced 3 weeks An atovaquone/proguanil donation program in Kenya and after a directly observed standard treatment course of ato- Uganda implemented in 1999 (and discontinued in 2001) [28] vaquone/proguanil. High-performance liquid chromatography gave rise to concerns that atovaquone/proguanil needs to be (Shimatzu) on day 2 of treatment confirmed a drug concen- protected, because new, safe, and affordable antimalarial drugs tration of atovaquone above the required therapeutic plasma are unlikely to be developed in the near future [29]. Protection can be achieved by judicious use and by combining atovaquone/ All patients were retreated successfully either with coartem- proguanil with artemisinin derivates, as is practiced in Thailand ether (artemether and lumefantrine; patients B, C, and D), other [30]. But the combination of atovaquone/proguanil with ar- artemisinin-based combinations (patient E), or mefloquine (pa- temisinin derivates will be largely unaffordable in the devel- tient A). Except for patient B, PCR-RFLP revealed wt in codon 1544 • JID 2004:190 (1 November) • Wichmann et al.
The present study was performed to screen imported isolates weeks after atovaquone/proguanil therapy, with several negative of P. falciparum for mutations previously reported to be as- blood smears in between. Reinfection can be excluded in 4 sociated with in vivo atovaquone/proguanil resistance. In 504 cases, because therapy was initiated in a European country. The samples, 495 were successfully tested for Tyr268Ser and 391 for other patient left the endemic area 8 days after the last dose Tyr268Asn on codon 268 of the parasite’s cyt b gene. One of of atovaquone/proguanil was received. Considering the intra- these mutations (Tyr268Ser) was detected. The fact that 2 inde- hepatic efficacy of atovaquone, it does not seem likely that the pendent PCRs were used to reveal the presence of the Tyr268- patient was reinfected during that time.
Ser mutation, whereas only 1 focused on the Asn268 mutation,explains the different success rates of the testing. Technical as- CONCLUSION
pects of the PCR protocols might also be responsible for this This type of molecular surveillance has little effect on treatment discrepancy. Given the size of our sample, it can be derived decisions for the individual travelers returning from regions with a 5% probability of error that the prevalence of Tyr268Ser where falciparum malaria is endemic. The high failure rate of in the European patient pool is !0.96% and that the prevalence atovaquone/proguanil in our population may be attributed to overreporting. Nevertheless, careful treatment follow-ups are In our subsample, 5 (8.62%) of 58 patients had atovaquone/ recommended after therapy with all antimalarial drugs. Codon- proguanil treatment failure. Compared with a large study per- 268 mutations located on the parasite’s cyt b gene seem to be formed in Thailand (n p 530), where failures occurred in 2.8% a sufficient, but not a necessary, cause for atovaquone/proguanil of patients treated with atovaquone/proguanil alone, this is an treatment failure. Further studies are recommended to elucidate unexpectedly high number [31]. Because the reporting within the mechanisms of resistance development and drug failure.
our network is self-selected, it might be assumed that treatment When used within a large clinical network, screening for failures are more likely to be reported (and subsequently over- molecular resistance markers in travelers with falciparum ma- represented) in our population. Therefore, this number must laria has an unsurpassed advantage. When efficient methods be interpreted with caution. Poor compliance with the treat- and reliable molecular markers are available, data on their prev- ment regimen can be excluded in all 5 cases. Impaired bio- alence can be used as an early warning system for changes availability of the drug could serve as another explanation of occurring in endemic areas, thus providing additional infor- recrudescence. For atovaquone, concomitant food intake plays mation that may be crucial for regional and international drug- an important role, because of a food-induced increase in drug policy changes. With regard to the 2 codon-268 mutations, solubility and, hence, bioavailability [32]. The drug concentra- Tyr268Ser and Tyr268Asn, which have previously been de- tion of atovaquone has been measured (and was sufficient) in scribed in patients with atovaquone/proguanil treatment fail- only 1 of the patients (patient E). In this case, the P. falciparum ure, we can be 95% confident that the current prevalence in isolate did not carry any codon-268 mutation. According to the European patient pool is !0.96% and !0.77%, respectively.
our data, Tyr268Ser seems to be a sufficient, but not a necessary, To the best of our knowledge, this is the first large-scale study cause for atovaquone/proguanil treatment failure—it was pres- on the prevalence of codon-268 mutations in a series of im- ent in only 1 of 5 patients in the atovaquone/proguanil failure ported P. falciparum isolates and is the first attempt to quantify group but in none of the successfully treated patients. Because the association between the prevalence of these mutations and Tyr268Asn was ruled out as a cause of the observed atovaquone/ atovaquone/proguanil treatment failure.
proguanil failures, it might be assumed that other mechanismsor other mutations on the cyt b gene might contribute to thedevelopment of resistance, in addition to the previously de- Acknowledgment
scribed codon-268 mutations. However, in 4 of 5 isolates, all We thank all site staff, who have been invaluable locally in collecting mutations previously described as involved in atovaquone re- sistance in vivo and in vitro [16] were ruled out by sequencinga 716-bp fragment of the cyt b gene [24].
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1546 • JID 2004:190 (1 November) • Wichmann et al.

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