The study focused on the identification and utilization of 18 S ribosomal RNA sequences as the basis of detection of different species of malarial parasites. 24 different P.falciparum specific oligonucleotide probes were synthesized based on rRNA sequences comparison. These were further screened for specificity by hybridization with total RNA isolated from P.falciparum. P. knowlesi and Humans. A mixture of 16 hybridization specific P.falciparum probes could detect upto 0.00039 % parasitaemia using total RNA extracted form parasite dilutions in uninfected human blood. A Plasmodium genus conserved probe and two P.vivax specific probes were also evaluated. 

 

In a limited field setting 91 samples were analysed using different probes. A higher level of sensitivity and specificity was obtained in comparision to conventional microscopy. Most significant was the detection of mixed infection cases, which has been missed by microscopy. 

 

To further adapt the procedure for field use a single step method of cell lysis and immobilization of total RNA was formulated using a mixture of 10 % methanol and 1% Tween 20. Initial studies on adaptation of the technique for non-isotopic detection demonstrated limits of 0.01 % parasitaemia using a single alkaline phosphatase labelled probe. 

 

Based on the sequence diversity of the Plasmodium 18 S rRNA, oligonucleotide primers were designed to give different size fragments for P.falciparum and P.vivax in a PCR. The primers were chosen such that the 5' primer was Plasmodium conserved while the 3' primers were species specific. The procedure yielded 1.4 kb and 0.5 kb fragments for P.falciparum and P.vivax respectively. Limited field-testing of this procedure demonstrated the utility of a ribosomal gene based species-specific malaria diagnosis.