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

Abstract

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.

Conclusion: 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.

Figure 1

Restriction digest method for detecting codon 268 mutations of cytochrome bc1 A: 384 bp amplification product with the primer pair CYTb3/CYTb5, digested by NsiI, K1 wild type (TAT) and AAT mutation (NGATV01) are cut (359 bp); B: 171 bp amplification product with the primer pair CYTb2/CYTb6, digested by AlwNI, only TN352 with the TCT mutation is cut to a 147 bp product; C: 174 bp amplification product with the primer pair CYTb2/CYTb7, digested by SspI, TAT wild type (K1) and TCT mutation (TN352) are digested (150 bp), while AAT mutation (NGATV01) remains uncut.