Background
The rapid emergence of resistance to standard antimalarial drugs has become a serious global health problem in endemic countries. For affected travellers returning to industrialised countries, effective treatment is available and resistance is as yet not a frequent problem in the treatment of falciparum malaria. The recently introduced drug Malarone
® is a combination of atovaquone and proguanil and is used for treatment and prophylaxis. There is certain evidence that parasites may quickly develop resistance to atovaquone and proguanil. When treated with atovaquone alone, one study showed that 33% of patients experienced recrudescence of parasitaemia [
1]. In combination with proguanil, cure rates from 99–100% were achieved [
2‐
7]. In 2002, the first case of
in vivo resistance to atovaquone and proguanil in a non-immune European traveller, returning from Nigeria, was reported [
8].
Atovaquone acts by inhibiting mitochondrial electron transport [
9] and collapsing mitochondrial membrane potential [
10]. It has been suggested that atovaquone, based on its structural similarity to ubiquinol, binds to the parasitic cytochrome bc
1 [
11]. Mutations in the cytochrome bc
1 gene of the parasite mitochondrial genome have been described as confering atovaquone resistance. Two mutations in
Pneumocystis carinii at the ubiquinol-binding pocket (Q
0 domain) are associated with atovaquone prophylaxis failure [
12]. In
in vitro resistant
Toxoplasma gondii lines two mutations at codon 129 and 254 were found to confer atovaquone resistance. Atovaquone-resistant
Plasmodium yoelii lines have been derived by sub-therapeutic treatment of infected mice. Five mutations near the putative atovaquone binding pocket have been identified, including a substitution of tyrosine by cysteine at codon 268 [
13]. In a similar study three mutations at the cytochrome b gene of atovaquone resistant
Plasmodium berghei lines were found to be associated with resistance to atovaquone. The mutations at codon 133 or 144, in addition to an amino acid change at codon 284, led to increased resistance levels.[
14]. Atovaquone resistant lines of
Plasmodium falciparum have been derived
in vitro by surviving various concentrations [
15]. An initial mutation at codon 133 was found to confer low resistance, which could be increased by an additional mutation in the domain from codon 272 to codon 280.
In vivo the cytochrome bc
1 sequence of a
P. falciparum isolate from a Thai patient with recrudescence after atovaquone and pyrimethamine treatment showed a mutation at codon 268 resulting in the substitution of tyrosine by serine [
1,
15]. An amino acid change to asparagine at the same codon was described in an English patient travelling to Nigeria who failed atovaquone/proguanil therapy [
8].
In this report we describe laboratory derived mutations of the cytochrome bc1 gene of P. falciparum after sub-curative administration of atovaquone alone or in combination with cycloguanil. These in vitro changes have been compared with mutations of an in vivo isolate derived from a patient with recrudescence after atovaquone/proguanil treatment. Based on this information we developed a novel PCR-RFLP method for the detection of mutations at codon 268, associated with resistance to atovaquone/proguanil.
Discussion
Atovaquone is an antimalarial substance that leads to an effective clearance of initial parasitaemia, but shows high recrudescence rates when used in monotherapy [
1]. In order to improve the therapeutic response and slow the development of drug resistance atovaquone is used in a fixed combination with proguanil (Malarone
®). Atovaquone acts by collapsing the mitochondrial membrane potential and therefore inhibiting parasite respiration [
10]. Single point mutations at the cytochrome bc
1 gene of
P. falciparum have been associated with atovaquone resistance [
15]. We investigated sequence changes on the
P. falciparum cytochrome bc
1 gene after exposure to atovaquone alone and in combination with cycloguanil, the active metabolite of proguanil. Cultures that survived atovaquone concentrations from 2 × 10
-8 up to 10
-7 M alone or in combination with cycloguanil showed a single point mutation at codon 133 (ATG to ATT) resulting in an amino acid change from methionine to isoleucine (M133I). When subjected to 2 × 10
-7 M atovaquone in the culture medium, the parasites showed an additional mutation at codon 271 (TTA to TTC) resulting in an amino acid change from leucine to phenylalanine (L271F). No second mutation was found for parasites that survived 2 × 10
-7 M atovaquone in combination with cycloguanil. The M133I mutation has also been reported from an atovaquone resistance line of
P. berghei [
14] but caused by a change from ATG to ATA. A mutation at codon 271 (TTA to GTA) has been described already for atovaquone resistant
P. yoelii lines (L271V) in combination with the K272R mutation [
13]. We found the mutation L271F in addition to the M133I change. Tandem mutations composed of M133I (ATG to ATA) with a second change in the region of codon 272 to 280 at the cytochrome b gene have been reported by Korsinczky et al. [
15] after similar
in vitro selection of mutations. Similar to our findings, he described an initial mutation at codon 133 resulting in a steric alteration at the putative atovaquone binding site. While the different tandem mutations do not affect predicted binding sites, they may enhance the alteration by the 133 substitution.
As we could demonstrate, the treatment of the K1 parasite culture with both drugs at sub-curative dosages did not prevent the development of the M133I mutation. Since the laboratory strain K1 is resistant to cycloguanil, a protective effect of the combination can be expected at higher doses of cycloguanil and has been shown for 2 × 10
-7 M atovaquone in combination with cycloguanil. The influence of proguanil on the development of resistance to Malarone
® in vitro has not been investigated. As in vitro study showed, acts proguanil compared to cycloguanil by distinct activities and has a synergistic effect in combination with atovaquone [
18,
19].
Sequencing of a
P. falciparum clone derived from an
in vivo treatment failure with Malarone
® detected a single point mutation at codon 268 of the cytochrome b gene. The change of TAT to TCT resulted in an amino acid substitution from tyrosine to serine (Y268S). The same mutation has been described before in a Thai patient with recrudescence after atovaquone and pyrimethamine treatment and a 10,000 fold increase of IC
50 to atovaquone [
1,
15]. Fivelman et al. [
8] showed a case of Malarone
® resistance
in vivo with 800 fold increase in IC
50 to atovaquone and a change to AAT at codon 268 resulting in a substitution to asparagine (Y268N). Furthermore the Y268S mutation has also been identified in blood samples of a
P. falciparum infected traveller returning from Cameroon to Denmark who failed Malarone
® therapy (personal communication, Kim David). In all cases, the substitution of the bulky Tyr268 by less bulky amino acids in the region of the ubiquinol oxidation site might affect the fit and binding of the drug.
The mutation at codon 133, although observed
in vitro, apparently does not influence development of
in vivo resistance to atovaquone/proguanil. However, the three cases of confirmed atovaquone/proguanil resistance came from West Africa where Malarone
® is not widely used yet. Increasing drug pressure with atovaquone/proguanil in endemic areas may also select for the M133I mutation. Investigations on atovaquone resistant K1 clones
in vitro found for the double amino acid mutation (M133I and G280D) 5 to 9% loss of fitness compared to the sensitive clone, but no detectable loss of fitness for the single mutation M133I [
18].
For the development of a diagnosis test for resistance to atovaquone/proguanil we focused on the two mutations at codon 268 of the parasite cytochrome bc1. Out of four molecular analysed treatment failures to date with either atovaquone/proguanil or atovaquone/pyrimethamine, all four samples showed mutations at codon 268. Both amino acid changes, Y268N and Y268S, resulted in extremely high increase of the IC50. Despite the low number of cases described to date indicate these results the relevance of codon 268 polymorphisms as potential resistance marker. To achieve a higher sensitivity, a nested PCR was designed with 3 different seconds rounds. Primers that anneal near the target mutations have been changed in one or two nucleotides in order to create restriction sites. The TAT wild type and the TCT mutation is demonstrated by the direct cut of the PCR product, while the AAT mutation is indicated when SspI does not cut. This might be a disadvantage of the method. However, with consequent use of positive and negative controls this is a precise method for the detection of codon 268 mutations.
Authors' contributions
BS carried out the in vitro cultivation of P. falciparum as well as the molecular genetic studies and drafted the protocol. MA participated in design and coordination of the molecular genetic studies. AS participated in cloning, sequencing and sequence alignment. TJ conceived of the study and participated in its coordination. All authors read and approved the final manuscript.