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Antimicrobial Agents and Chemotherapy, April 2009, p. 1722-1723, Vol. 53, No. 4
0066-4804/09/$08.00+0     doi:10.1128/AAC.01427-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

LETTER TO THE EDITOR

Plasmodium falciparum and Dihydrofolate Reductase I164L Mutations in Africa

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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.

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1. Alker, A. P., V. Mwapasa, and S. R. Meshnick. 2004. Rapid real-time PCR genotyping of mutations associated with sulfadoxine-pyrimethamine resistance in Plasmodium falciparum. Antimicrob. Agents Chemother. 48:2924-2929.[Abstract/Free Full Text]
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2. Alker, A. P., V. Mwapasa, A. Purfield, S. J. Rogerson, M. E. Molyneux, D. D. Kamwendo, E. Tadesse, E. Chaluluka, and S. R. Meshnick. 2005. Mutations associated with sulfadoxine-pyrimethamine and chlorproguanil resistance in Plasmodium falciparum isolates from Blantyre, Malawi. Antimicrob. Agents Chemother. 49:3919-3921.[Abstract/Free Full Text]
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3. Francis, D., S. L. Nsobya, A. Talisuna, A. Yeka, M. R. Kamya, R. Machekano, C. Dokomajilar, P. J. Rosenthal, and G. Dorsey. 2006. Geographic differences in antimalarial drug efficacy in Uganda are explained by differences in endemicity and not by known molecular markers of drug resistance. J. Infect. Dis. 193:978-986.[CrossRef][Medline]
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4. Juliano, J. J., P. Trottman, V. Mwapasa, and S. R. Meshnick. 2008. Detection of the dihydrofolate reductase-164L mutation in Plasmodium falciparum infections from Malawi by heteroduplex tracking assay. Am. J. Trop. Med. Hyg. 78:892-894.[Abstract/Free Full Text]
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5. Livak, K. J. 1999. Allelic discrimination using fluorogenic probes and the 5' nuclease assay. Genet. Anal. 14:143-149.[Medline]
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6. Lynch, C., R. Pearce, H. Pota, J. Cox, T. A. Abeku, J. Rwakimari, I. Naidoo, J. Tibenderana, and C. Roper. 2008. Emergence of a dhfr mutation conferring high-level drug resistance in Plasmodium falciparum populations from Southwest Uganda. J. Infect. Dis. 197:1598-1604.[CrossRef][Medline]
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7. Ochong, E., D. J. Bell, D. J. Johnson, U. D'Alessandro, M. Mulenga, S. Muangnoicharoen, J. P. Van Geertruyden, P. A. Winstanley, P. G. Bray, S. A. Ward, and A. Owen. 2008. Plasmodium falciparum strains harboring dihydrofolate reductase with the I164L mutation are absent in Malawi and Zambia even under antifolate drug pressure. Antimicrob. Agents Chemother. 52:3883-3888.[Abstract/Free Full Text]
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8. Paget-McNicol, S., and A. Saul. 2001. Mutation rates in the dihydrofolate reductase gene of Plasmodium falciparum. Parasitology 122:497-505.[Medline]

						Alisa P. Alker Jonathan J. Juliano Steven R. Meshnick* University of North Carolina School of Medicine and School of Public Health CB#7435 Chapel Hill, North Carolina 27599-7435
						* Phone: (919) 966-7414, Fax: (919) 966-2089, E-mail: meshnick{at}unc.edu

Authors' Reply

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We thank Dr. Alker and colleagues for raising some interesting issues regarding our article (6) and for the opportunity to clarify any misunderstandings.

We are grateful for a more robust description of controls. However, we remain a little confused about how the inclusion of standard curves of plasmid DNA in the plate (even if included in every run) prevents problems associated with cross-reacting probes for individual reactions, with variable isolate DNA concentrations. The authors are correct that the performance of real-time assays can depend on the cycler, reagents, and batch of probe. However, we observed specificity and variability problems in three real-time PCR cyclers (Bio-Rad Chromo 4, MJ Research Opticon 2, and ABI Prism 7700 system) and with multiple batches of probe. Conversely, our optimized assay was found to be accurate and precise in all three systems, irrespective of the batch of probe used.

We strongly disagree that our method was incompletely validated. As stated in our original article (see Materials and Methods in reference 6), the same Malawian samples were previously genotyped by allele-specific restriction analysis, by which the DHFR-164 mutation was also undetectable (2). We agree that whole-genome amplification (WGA) may change haplotype frequencies. However, we were assessing the allele rather than the intact haplotype. Perhaps of more concern in this context is allele dropout, which sometimes arises from an imbalanced efficiency between alleles and can result in one allele being preferentially amplified in WGA (7). However, this is unlikely to have occurred, because WGA was also necessary for eight (21%) of the Thai samples and six of these were positive for the DHFR-164 mutant allele. In the unlikely event that allele dropout did occur, this would not affect our interpretation, since only 22 (13.9%) of the Malawian cohort and 11 (26%) of the Zambian cohort had <10,000 copies of parasite DNA per microliter and were thus subjected to WGA.

The authors infer that we understated the body of evidence supporting the emergence of the DHFR-164L mutation in Africa and cite three additional articles to support this (3-5). However, the article by Juliano et al. with the associated heteroduplex tracking assay was cited in the introduction of our article. All newly emerging technologies have inherent limits of sensitivity and specificity. Currently, therefore, the most convincing data for the emergence of this allele in Africa are those described by Lynch et al., who utilized more traditional methodologies (5). That article was published after we submitted our article to Antimicrobial Agents and Chemotherapy, and both the additional studies (3, 5) were conducted in Uganda, not Malawi (or Zambia). Nonetheless, the regional selection hypothesis is certainly interesting and worthy of further study.

Finally, as discussed in our original article, we completely concur that there are differences between the patients in our study and those in the study by Alker et al. (see Discussion in reference 6), and neither were sampled to represent the general population.

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1. Alker, A. P., V. Mwapasa, and S. R. Meshnick. 2004. Rapid real-time PCR genotyping of mutations associated with sulfadoxine-pyrimethamine resistance in Plasmodium falciparum. Antimicrob. Agents Chemother. 48:2924-2929.[Abstract/Free Full Text]
2
2. Bell, D. J., S. K. Nyirongo, M. Mukaka, E. E. Zijlstra, C. V. Plowe, M. E. Molyneux, S. A. Ward, and P. A. Winstanley. 2008. Sulfadoxine-pyrimethamine-based combinations for malaria: a randomised blinded trial to compare efficacy, safety and selection of resistance in Malawi. PLoS ONE. 3:e1578.[CrossRef][Medline]
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3. Francis, D., S. L. Nsobya, A. Talisuna, A. Yeka, M. R. Kamya, R. Machekano, C. Dokomajilar, P. J. Rosenthal, and G. Dorsey. 2006. Geographic differences in antimalarial drug efficacy in Uganda are explained by differences in endemicity and not by known molecular markers of drug resistance. J. Infect. Dis. 193:978-986.[CrossRef][Medline]
4
4. Juliano, J. J., P. Trottman, V. Mwapasa, and S. R. Meshnick. 2008. Detection of the dihydrofolate reductase-164L mutation in Plasmodium falciparum infections from Malawi by heteroduplex tracking assay. Am. J. Trop. Med. Hyg. 78:892-894.[Abstract/Free Full Text]
5
5. Lynch, C., R. Pearce, H. Pota, J. Cox, T. A. Abeku, J. Rwakimari, I. Naidoo, J. Tibenderana, and C. Roper. 2008. Emergence of a dhfr mutation conferring high-level drug resistance in Plasmodium falciparum populations from southwest Uganda. J. Infect. Dis. 197:1598-1604.[CrossRef][Medline]
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6. Ochong, E., D. J. Bell, D. J. Johnson, U. D'Alessandro, M. Mulenga, S. Muangnoicharoen, J. P. Van Geertruyden, P. A. Winstanley, P. G. Bray, S. A. Ward, and A. Owen. 2008. Plasmodium falciparum strains harboring dihydrofolate reductase with the I164L mutation are absent in Malawi and Zambia even under antifolate drug pressure. Antimicrob. Agents Chemother. 52:3883-3888.[Abstract/Free Full Text]
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7. Peng, W., H. Takabayashi, and K. Ikawa. 2007. Whole genome amplification from single cells in preimplantation genetic diagnosis and prenatal diagnosis. Eur. J. Obstet. Gynecol. Reprod. Biol. 131:13-20.[CrossRef][Medline]

						Andrew Owen* Department of Pharmacology and Therapeutics 70 Pembroke Place University of Liverpool, Liverpool L69 3GF, United Kingdom Edwin Ochong David J. Bell David J. Johnson Liverpool School of Tropical Medicine Pembroke Place Liverpool L35QA, United Kingdom Umberto d'Alessandro Department of Parasitology Prince Leopold Institute of Tropical Medicine Antwerp, Belgium Modest Mulenga Tropical Diseases Research Center (TDRC) Ndola, Zambia Sant Muangnoicharoen Liverpool School of Tropical Medicine Pembroke Place Liverpool L35QA, United Kingdom J. P. Van Geertruyden Department of Parasitology Prince Leopold Institute of Tropical Medicine Antwerp, Belgium Peter A. Winstanley School of Clinical Sciences University of Liverpool Liverpool, United Kingdom Patrick G. Bray Stephen A. Ward Liverpool School of Tropical Medicine Pembroke Place Liverpool L35QA, United Kingdom
						* Phone: 44 (0) 151 794 5919 Fax: 44 (0) 151 794 5656 E-mail: aowen{at}liv.ac.uk

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Antimicrobial Agents and Chemotherapy, April 2009, p. 1722-1723, Vol. 53, No. 4
0066-4804/09/$08.00+0     doi:10.1128/AAC.01427-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Alker, A. P. Articles by Ward, S. A. Search for Related Content PubMed PubMed Citation Articles by Alker, A. P. Articles by Ward, S. A.