TY - JOUR
T1 - Critical comparison of molecular genotyping methods for detection of drug resistant Plasmodium falciparum
AU - Ranford-Cartwright, L. C.
AU - Johnston, K. L.
AU - Abdel-Muhsin, A. M.
AU - Khan, B. K.
AU - Babiker, H. A.
N1 - Funding Information:
Acknowledgements This work was funded by the International Atomic Energy Agency (IAEA), Austria, and the UK Medical Research Council. A. Abdel-Muhsin is supported by a WI-IOiTDR training award and the Gordon Memorial College Trust Fund, UK. We thank those attending the IAEA Regional Training Course RAF61025, Kampala, Uganda, in September 2001, for their feedback on these results and their encouragement to publish them. Detailed protocols are available to download at http:l/www.gla.ac.uklibls/IIllrc/protocols.htm References Abdel-Muhsin, A.-M. A., Ranford-Cartwright, L. C., Medani, A.-R., Ahmed, S., Suleiman, S., Khan, B., Hunt, I’., Wal-liker. D. & Babiker. H. A. (2002). Detection of mutations in the Plasmodium falciparum dihydrofolate reductase (dhfi) gene by dot-blot hybridization. American Journal of Tropical Medicine and Hygiene. 67,24-27. De Pecoulas, I’. E., Basco, L. K., Abdallah, B., Dje, M. K., Le Bras, J. & Mazabraud, A. (1995). Plasmodium falcipanrm: detection of antifolate resistance by mutation-specific re- striction enzyme digestion. Experimental Parasitology, 80, 483-487. Duraisingh, M. T., Curtis, J. & Warhurst, D. C. (1998). Plasmodium falciparum: detection of polymorphisms in the dihydrofolate reductase and dihydropteroate synthetase genes by PCR and restriction digestion. Experimental Para-sitoloat. -,-89. ,-l-8. ~- Fidock, D. A., Nomura, T., Talley, A. K., Cooper, R. A., Dzekunov, S. M., Ferdia, M. T., Ursos. L. M. B.. Sidhu. A. B. S., Naude, B.,.Deitsch, K. W:, Su, X. Z., Woo&on, J.C., Roepe, P. D. & Wellems, T. E. (2000). Mutations in the I? falcipanrm digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance. Mole-cular Cell, 6, 861-871. Lambros, C. & Vanderberg, J. P. (1979). Synchronization of Plasmodiumf alciparum erythrocytic stages in culture. ~oumal of Parasitology, 65, 418-420. Peterson, D. S., Walliker, D. & Wellems, T. E. (1988). Evidence that a point mutation in dihydrofolate reductase-thymidylate synthase confers resistance to pyrimethamine in falciparum malaria. Proceedingso f the National Academv _ of I Scienceso fthe USA. 85.9114-91 IS. Plowe, C. V., Djimde, A., Bouare, M., Doumbo, 0. & Well-ems, T. E. (1995). Pyrimethamine and proguanil resis-tance-conferring mutations in Plasmod&m-falciparum dihvdrofolate reductase: oolvmerase chain reaction methods for surveillance in Africa: American Journal of Tropical Medi-cine and Hygiene, 52, 565-568. Shaio, M. F., Wang, I’., Lee, C. S., Sims, I’. F. G. & Hyde, J. E. (1998). Develooment and comoarison of auantitative assays for the dihydropteroate synthetase codon’ muta-tion associated with sulfadoxine resistance in Plasmodium falciparum. Parasitology, 116, 203-210. Thaithong, S., Beale, G. H., Fenton, B., McBride, J., Rosario, V., Walker, A. & Walliker, D. (1984). Clonal diversity in a single isolate of the malaria parasite Plasmodiumf alciparum. Transactions of the Royal Society of Tropical Medicine and Hvgiene. X242-245. Trager, We.& Jensen, J. B. (1976). Human malaria parasites in continuous culture. Science, 193, 673-675. Wang, I’., Brooks, D. R., Sims, P. F. G. & Hyde, J. E. (1995). A mutation-soecitic PCR svstem to detect seauence varia-tion in the dihydropteroath synthetase gene of Plasmodium falciparum. Molecular and Biochemical Parasitology, 71, 115-125. Wang, P., Read, M., Sims, P. F. G. & Hyde, J. E. (1997). Sulfadoxine resistance in the human malaria parasite Plas-modiumf alciparum is determined by mutations in dihydro-pteroate synthetase and an additional factor associated with folate utilization. Molecular Microbioloav. 23, 979-986. WHO (1996). Assessmento f therapeu&&a~y of antimalarial drugsf or uncomplicatedfalciparumm alaria in areas with intense transmission. Geneva: World Health Organization, mimeo-graphed document WHOMAL96.1077. Zolg, J. W., Chen, G.-X. & Plitt, J. R. (1990). Detection of pyrimethamine resistance in Plasmodium falciparum by mu-tation-specific polymerase chain reaction. Molecular and Biochemical Parasitology, 39, 257-266.
PY - 2002
Y1 - 2002
N2 - We have critically evaluated 3 techniques for the detection of mutations conferring drug resistance of P1asmodium falciparum, using samples containing known numbers of well-characterized parasites and artificial mixtures of these parasites at known proportions. We compared the sensitivity and specificity of mutation-specific polymerase chain reaction (MS-PCR), polymerase chain reaction followed by restriction enzyme digestion at polymorphic sites (PCR/RFLP), and a dot-blot/probe hybridization technique, for detection of point mutations at nucleotide 323 of the P. falciparum dihydrofolate reductase gene (dhfr) that confer resistance to the antimalarial drug pyrimethamine. We have also investigated the benefits in terms of sensitivity and reproducibility of the incorporation of radiolabelled nucleotides into the PCR/RFLP assay. We found that MS-PCR was very sensitive-at least 10 parasites could be detected in a sample-but non-specific amplification resulted in erroneous typing of some samples. PCR/RFLP was less sensitive; 10 parasites per sample could not always be detected, but the technique was specific. The addition of radiolabelled nucleotides to the assay did not markedly improve the sensitivity but the results were easier to read and there was less subjectivity in scoring the results. The dot-blot/probe hybridization technique was specific and sensitive, with similar levels of specificity and sensitivity to PCR/RFLP. On balance, the dot-blot/probe hybridization technique seems best suited to large-scale epidemiological surveys of genes associated with antimalarial drug resistance.
AB - We have critically evaluated 3 techniques for the detection of mutations conferring drug resistance of P1asmodium falciparum, using samples containing known numbers of well-characterized parasites and artificial mixtures of these parasites at known proportions. We compared the sensitivity and specificity of mutation-specific polymerase chain reaction (MS-PCR), polymerase chain reaction followed by restriction enzyme digestion at polymorphic sites (PCR/RFLP), and a dot-blot/probe hybridization technique, for detection of point mutations at nucleotide 323 of the P. falciparum dihydrofolate reductase gene (dhfr) that confer resistance to the antimalarial drug pyrimethamine. We have also investigated the benefits in terms of sensitivity and reproducibility of the incorporation of radiolabelled nucleotides into the PCR/RFLP assay. We found that MS-PCR was very sensitive-at least 10 parasites could be detected in a sample-but non-specific amplification resulted in erroneous typing of some samples. PCR/RFLP was less sensitive; 10 parasites per sample could not always be detected, but the technique was specific. The addition of radiolabelled nucleotides to the assay did not markedly improve the sensitivity but the results were easier to read and there was less subjectivity in scoring the results. The dot-blot/probe hybridization technique was specific and sensitive, with similar levels of specificity and sensitivity to PCR/RFLP. On balance, the dot-blot/probe hybridization technique seems best suited to large-scale epidemiological surveys of genes associated with antimalarial drug resistance.
KW - Dihydrofolate
KW - Drug resistance
KW - Genotyping techniques
KW - Malaria
KW - Plasmodium falciparum
KW - Pyrimethamine
KW - Reductase
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U2 - 10.1016/S0035-9203(02)90446-3
DO - 10.1016/S0035-9203(02)90446-3
M3 - Article
C2 - 12474492
AN - SCOPUS:0036759146
SN - 0035-9203
VL - 96
SP - 568
EP - 572
JO - Transactions of the Royal Society of Tropical Medicine and Hygiene
JF - Transactions of the Royal Society of Tropical Medicine and Hygiene
IS - 5
ER -