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Antimicrobial Agents and Chemotherapy, February 2004, p. 466-472, Vol. 48, No. 2
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.2.466-472.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Genotyping of Plasmodium falciparum Pyrimethamine Resistance by Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry

Florian Marks,¹ Christian G. Meyer,¹ Jürgen Sievertsen,¹ Christian Timmann,¹ Jennifer Evans,² Rolf D. Horstmann,¹ and Jürgen May^1*

Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany,¹ Kumasi Centre for Collaborative Research in Tropical Medicine, Kumasi, Ghana²

Received 26 August 2003/ Returned for modification 9 October 2003/ Accepted 24 October 2003

Increasing resistance, recrudescences, and treatment failure have led to the replacement of chloroquine with the combination of pyrimethamine (PYR) and sulfadoxine (SDX) as the first-line antimalarial drugs for treatment of uncomplicated Plasmodium falciparum malaria in several areas where this disease is endemic. The development of resistance to PYR-SDX is favored by incomplete treatment courses or by subtherapeutic levels in plasma. PYR-SDX resistance has been associated with several single-nucleotide polymorphisms (SNPs) in the P. falciparum dihydrofolate reductase (pfdhfr) and the P. falciparum dihydropteroate synthetase (pfdhps) genes. We have established assays based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) that conveniently allow the identification of SNPs associated with PYR resistance. Variants occurring at codon positions 16, 51, 59, and 108 of the pfdhfr gene were analyzed by MALDI-TOF MS in synthetic oligonucleotides to determine the detection threshold. In addition, 63 blood samples from subjects with P. falciparum parasitemia of various degrees were analyzed. The results were compared to those obtained by DNA sequencing of the respective gene fragment. The results of MALDI-TOF MS and DNA sequencing were consistent in 40 samples. In 23 samples two or three pfdhfr variants were detected by MALDI-TOF assays, whereas DNA-sequencing revealed one variant only. Simultaneous detection of two different mutations by biplex assays was, in principle, feasible. As demonstrated by the example of PYR resistance, MALDI-TOF MS allows for rapid and automated high-throughput assessment of drug sensitivity in P. falciparum malaria.

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* Corresponding author. Mailing address: Bernhard Nocht Institute for Tropical Medicine, Department of Molecular Medicine, Bernhard-Nocht-Str. 74, 20359 Hamburg, Germany. Phone: 49-40-42818-369. Fax: 49-40-42818-512. E-mail: may{at}bni.uni-hamburg.de.

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Antimicrobial Agents and Chemotherapy, February 2004, p. 466-472, Vol. 48, No. 2
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.2.466-472.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Marks, F., Evans, J., Meyer, C. G., Browne, E. N., Flessner, C., von Kalckreuth, V., Eggelte, T. A., Horstmann, R. D., May, J. (2005). High Prevalence of Markers for Sulfadoxine and Pyrimethamine Resistance in Plasmodium falciparum in the Absence of Drug Pressure in the Ashanti Region of Ghana. Antimicrob. Agents Chemother. 49: 1101-1105 [Abstract] [Full Text]