Design and validation of multiplex polymerase chain reaction as a diagnostic tool for species.

Background & Objectives: The highly sensitive method for a true understanding of malaria prevalence is of utmost importance for India's elimination strategy. The PCR reaction type with rapid detection, cost-effectiveness, and less workforce should be preferable. Multiplex PCR type accomplishes the present requirement by saving time and resources to find true surveillance data for malaria, especially in low-parasitemia/asymptomatic groups or populations.

Methods: The present study focuses on designing multiplex PCR (mPCR) to detect simultaneously Plasmodium genus (PAN) and two common Plasmodium species found in India. It is compared to standard nested PCR on 195 clinical samples to diagnose malaria. The mPCR was designed with a minimum number of primers, leading to less clogging and effective and enhanced detection. It contains one common reverse primer and three forward primers amplifying three targeted genes corresponding to P. falciparum, P. vivax, and Plasmodium genus.

Results: The sensitivity and specificity for mPCR were 94.06 and 95.74, respectively. The limit of detection for mPCR was 0.1 parasites/µl. The study has shown a ROC curve area for the mPCR of 0.949 for Plasmodium genus and P. falciparum and 0.897 for P. vivax with standard nPCR.

Interpretation & Conclusion: The mPCR is rapid in detecting species together, cost-effective, and requires fewer human resources than the standard nPCR. Therefore, the mPCR can be used as an alternative technique for the higher sensitive detection of the malaria parasite. It could also become a vital tool for determining malaria prevalence, facilitating the application of the most effective measures.