New molecular detection methods of malaria parasites with multiple genes from genomes

For the effective control of malaria, development of sensitive, accurate and rapid tool to diagnose and manage the disease is essential. In humans subjects, the severe form of malaria is caused by Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) and there is need to identify these parasites in acute, chronic and latent (during and post-infection) stages of the disease. In this study, we report a species specific and sensitive diagnostic method for the detection of Pf and Pv in humans. First, we identified intra and intergenic multiloci short stretch of 152 (PfMLS152) and 110 (PvMLS110) nucleotides which is present up to 44 and 34 times in the genomes of Pf and Pv respectively. We developed the single-step amplification-based method using isolated DNA or from lysed red blood cells for the detection of the two malaria parasites. The limit of detection of real-time polymerase chain reaction based assays were 0.1copyof parasite/μl for PfMLS152 and PvMLS110 target sequences. Next, we have tested 250 clinically suspected cases of malaria to validate the method. Sensitivity and specificity for both targets were 100% compared to the quantitative buffy coat microscopy analysis and real-time PCR (Pf-chloroquine resistance transporter (PfCRT) and Pv-lactate dehydrogenase (PvLDH)) based assays. The sensitivity of microscopy and real-time PCR (PfCRT and PvLDH primers) assays were 80.63%; 95%CI 75.22%-85.31%; p<0.05 and 97.61%; 95%CI 94.50%-99.21%; p<0.05 in detecting malaria infection respectively when compared to PfMLS152 and PvMLS110 targets to identify malaria infection in patients. These improved assays may have potential applications in evaluating malaria in asymptomatic patients, treatment, blood donors and in vaccine studies.