Malaria: use of restriction endonuclease digestion and mutation-specific PCR for antifolate resistance isolate...

Antifolate resistance isolates of Plasmodium falciparum in the blood of 56 patients was investigated by using PCR technology. DNA was extracted with three different methods from parasite lysate by phenol-chloroform, or from whole blood and from blood collected onto dry filter paper, by chelex-100. The expected 727-bp PCR product was obtained in all samples extracted by chelex-100, while three samples prepared by phenol-chloroform failed to show any amplified product. The crucial point mutation within the dhfr gene leading to pyrimethamine and cycloguanil resistance is localised in an Alul recognition site. Thus, the 727-bp PCR product was submitted to endonuclease digestion. Fifty out of the 56 blood samples analysed yielded the two expected restriction fragments and an undigested 727-bp band. These 50 samples likely represent mixed infection as also confirmed the specific mutation PCR. The six undigested samples amplify a 339-bp fragment using a nested PCR-specific for pyrimethamine resistance mutation. Our results show that, the rapid DNA extraction from blood using chelex-100 and the PCR endonuclease assay can be efficiently used for accurate chemosensitivity analysis in the field.