Measurement of minimum inhibitory concentration for malarial parasite Plasmodium falciparum by PCR technique

Abstract

Malaria remains an important problem of public health. Although the mortality and mobility are decreasing in some endemic areas but the problem of drug resistance especially in falciparum malaria against many of routinely used antimalarial drug has become serious. The assessment of Minimum Inhibitory Concentration (MIC) of drug susceptibility is therefore needed for monitoring and control malaria disease. In this study, the PCR technique has been applied for testing MIC value against 4 antimalarial drugs which are quinine, mefloquine, chloroquine and pyrimethamine. The tested MIC values from PCR has been compared with the MIC values derived from the conventional microscopic test. The results from the comparison of three DNA extraction methods, boiling method, phenol-chloroform extraction and DNA extraction kit, revealed that the standard phenol-chloroform method is the most suitable for this study. In addition, the RNA extraction kit is also suitable for RNA extraction. The extracted DNA and RNA has been tested for malaria parasite detection. The sensitivity of PCR with rap-1 primers is 0.05% which is slightly lower than the microscopic test whereas the sensitivity of RT-PCR is down to 0.01% as equal to the MIC from microscopic assay Moreover, the PCR with ssrRNA primers gave more sensitive result, down to 0.001% by both PCR and RT-PCR. The MIC against 4 drugs of pure clone P. falciparum, T9/94RC17, has been evaluated by using PCR and RT-PCR with rap-1 primers and the results showed the equal values to the microscopic assay except in mefloquine which has shown slightly difference. Beside, the modified direct PCR and RT-PCR with rap-1 primers has also been tested in T9/94RC17 but the techniques failed to meet the same standard. Finally, the assessment of MIC value by PCR with rap-1 primers in 3 wild isolates, MH20, TD12 and K60, had been performed and the promising results had been observed. From this study, the results revealed that PCR is an attractive alternative and has the potential for assessment of the MIC value especially for large scale screening.