Different mutation patterns of atovaquone resistance to Plasmodium falciparum in vitro and in vivo: rapid detection of codon 268 polymorphisms in the cytochrome b as potential in vivo resistance marker

Background: Resistance of Plasmodium falciparum to atovaquone in
vitro and in vivo has been associated to mutations in the parasite
cytochrome b gene. Methods: Cultures were sequentially subjected to
increasing doses of atovaquone alone or in combination with
cycloguanil and the cytochrome b gene was sequenced. Additionally,
we investigated the parasite cytochrome b gene of a patient
returning from Mali with Malarone(R) treatment failure in vivo.
Results: All strains that survived atovaquone concentrations in
vitro of 2 x 10(-8) to 2 x 10(7) M showed the M1331 mutation and
one strain with the highest atovaquone concentration the additional
mutation L171F. Sequencing of the in vivo treatment failure
revealed a point mutation at codon 268 resulting in an amino acid
change from tyrosine to serine. Based on the repeated emergence of
mutations at codon 268, but no detection of alterations at codon
133 in vivo, we developed a detection method for the diagnostic of
codon 268 polymorphisms as a potential atovaquone/proguanil
resistance marker. A nested PCR with 3 different pairs of primers
for the second round was designed. Each product was digested with
restriction enzymes, capable to distinguish the wild type from the
two reported mutations at codon 268. Conclusion: Mutations at codon
268 of the parasite cytochrome bc(1) gene are associated with
atovaquone/proguanil treatment failure in vivo and can be used as
potential resistance marker This method provides a novel and robust
tool to investigate the relevance of codon 268 polymorphisms as
resistance marker and to monitor the further emergence of
atovaquone/proguanil resistance.