Plasmodium falciparum and Dihydrofolate Reductase I164L Mutations in Africa

Abstract

Listen

Translate article

The paper by Ochong et al. modifies the real-time PCR method we published previously and applies it to samples from Malawi, Zambia, and Thailand (2, 7). These authors bring up several criticisms of the original technique, and we welcome this opportunity to clarify concerns about this method. We would also like to comment on some of their conclusions. The primary criticism of Ochong et al. of our assay of mutations of dihydrofolate reductase at position 164 (DHFR-164) is that there was nonspecific binding of the probes. We have previously reported this phenomenon (1), which is evident from the original paper describing the MGB probe (see Fig. 4 in reference 5). However, this background binding does not interfere with the assay's discriminating ability as long as proper controls (standard curves of both wild-type and mutant DNA at concentrations similar to the clinical samples) are included (1, 2). In addition, the assay's performance is dependent on the real-time PCR machine, reagents, and even the batch of probe (1). Thus, the assay's discriminating ability should be reoptimized when it is adapted to different conditions. Overall, we find the modified assay of Ochong et al. to be a successful adaptation of our assay to a new lab. However, this adaptation was incompletely validated. For example, the authors did not determine its sensitivity and specificity by running both real-time PCR and the gold standard allele-specific PCR with the same samples. Also, the authors should address the possibility that whole-genome amplification changes the frequencies of haplotypes from that in the original sample. There is a much stronger body of evidence supporting the emergence of the DHFR-164L mutation in Africa than is suggested in this paper. We recently confirmed the existence of the DHFR-164L mutation in Malawi by a heteroduplex tracking assay and direct sequencing (4). Furthermore, a subsequent report by Lynch et al. found the mutation in 14% of samples from southwestern Uganda collected in 2005 (6). Previously, in 2002, the mutation had been found at a prevalence of 1.25% in Kanungu in southwestern Uganda (3). This suggests that the mutation may be emerging regionally in Africa. Finally, it is important to note that all of the reported studies of the prevalence of the DHFR-164L mutation use patients enrolled in studies and were not sampled to represent the general population. Thus, small differences in prevalence between studies and study sites are to be expected.