Hostname: page-component-7c8c6479df-xxrs7 Total loading time: 0 Render date: 2024-03-28T05:07:54.631Z Has data issue: false hasContentIssue false

Development and comparison of quantitative assays for the dihydropteroate synthetase codon 540 mutation associated with sulfadoxine resistance in Plasmodium falciparum

Published online by Cambridge University Press:  01 March 1998

M.-F. SHAIO
Affiliation:
Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology (UMIST), PO Box 88, Manchester M60 1QD, UK Current address: Department of Parasitology and Tropical Medicine, National Defense Medical Center, PO Box 90048-506, Taipei, Taiwan, Republic of China.
P. WANG
Affiliation:
Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology (UMIST), PO Box 88, Manchester M60 1QD, UK
C.-S. LEE
Affiliation:
Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology (UMIST), PO Box 88, Manchester M60 1QD, UK
P. F. G. SIMS
Affiliation:
Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology (UMIST), PO Box 88, Manchester M60 1QD, UK
J. E. HYDE
Affiliation:
Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology (UMIST), PO Box 88, Manchester M60 1QD, UK

Abstract

A point mutation in codon 540 of the dihydropteroate synthetase (dhps) gene affecting sulfadoxine resistance has previously been found in parasites from patients with Plasmodium falciparum infection. Here, we investigated 4 methods of identifying this mutation in clinical specimens and established a reliable quantitative assay to estimate the percentage of resistant type in mixed infections. A diagnostic PCR assay based on allele-specific amplification was developed, which clearly typed the clinical specimens examined. The mutation in codon 540 introduces an additional FokI cleavage site which provided a second method to differentiate mutant from wild type, where the former gives rise to 2 characteristic fragments of 538 and 326 bp that are absent from the latter. To calibrate quantitatively the ratio of alleles in mixed samples, we constructed artificial mixes containing 2 plasmid DNAs, one carrying the mutation and the other a wild-type insert. When 32P-labelling was employed, the allele-specific PCR assay could detect the level of resistant type in a mixture down to 0·1–1%, while for the restriction enzyme/PCR analysis, the figure was approximately 10%. Furthermore, neither fluorescent dye-labelled terminator nor dye-labelled primer cycle sequencing was able to detect the mutant allele if it was present at less than 20–30%. We conclude that the allele-specific PCR assay is the most sensitive method of detecting the codon 540 mutation in P. falciparum dhps, and the method of choice for estimating the composition of mixed samples.

Type
Research Article
Copyright
1998 Cambridge University Press

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)