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
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 .
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 (email@example.com). 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 . 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 , 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 .
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-
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 . The
chondrial membrane electropotential . 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 , 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 . 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 . 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 .
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
. 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 .
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
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, 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
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 . 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) 
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 . 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
. 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-
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 . 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 . 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-
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  were ruled out by sequencinga 716-bp fragment of the cyt b gene . References
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Management eines Tabus ler Studien entsprechen (5, 6). (Sildenafil, Tadalafil, Vardenafil) suchenEXPECT-Studie: Therapie der erektilen DysfunktionErektionsstörung Hilfe in der hausärzt-Erektionsstörungen gehören zu den häufigsten sexuellenSchwierigkeiten, welche Patienten in der ärztlichen Praxis zurSprache bringen. In der Werbung und den Medien ist Sex einhälfte, von einer ED betrof
2:08-cv-02133-MPM-DGB # 152 Page 1 of 33 Thursday, 20 September, 2012 11:38:16 AM UNITED STATES DISTRICT COURT CENTRAL DISTRICT OF ILLINOIS URBANA DIVISION ____________________________________________________________________________ NECA-IBEW PENSION TRUST FUND, NECA-IBEW WELFARE TRUST FUND, and INTERNATIONAL BROTHERHOOD OF ELECTRICAL WORKERS LOCAL UNION Case No. 08-CV