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 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 M133I 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.


CONCLUSIONS
Mutations at codon 268 of the parasite cytochrome bc1 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.
Schwöbel
Babett
B
Department of Infectious Disease and Tropical Medicine, University of Munich, Leopoldstr, 5, 80802 Munich, Germany. babett.schwoebel@lrz.uni-muenchen.de
Alifrangis
Michael
M
Salanti
Ali
A
Jelinek
Tomas
T
D000818:Animals; D000962:Antimalarials / Q000378:metabolism / Q000627:therapeutic use; D000981:Antiprotozoal Agents / Q000378:metabolism / Q000627:therapeutic use; D053626:Atovaquone; D003062:Codon / Q000235:genetics*; D004351:Drug Resistance / Q000235:genetics*; D014450:Electron Transport Complex III / Q000235:genetics* / Q000378:metabolism; D005819:Genetic Markers / Q000235:genetics; D006801:Humans; D016778:Malaria, Falciparum / Q000175:diagnosis / Q000188:drug therapy / Q000378:metabolism; D009154:Mutation; D009285:Naphthoquinones / Q000378:metabolism* / Q000627:therapeutic use; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000201:enzymology* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length; D002727:Proguanil / Q000378:metabolism / Q000627:therapeutic use; D014227:Triazines / Q000378:metabolism / Q000627:therapeutic use
D000962:Antimalarials; D000981:Antiprotozoal Agents; D003062:Codon; D005819:Genetic Markers; D009285:Naphthoquinones; D014227:Triazines; C026009:cycloguanil; D014450:Electron Transport Complex III; D002727:Proguanil; D053626:Atovaquone