Validation of a Semiquantitative Real-Time PCR Assay for Malaria Diagnosis and Treatment Monitoring.

Microscopic examination of blood smears is the current gold standard for laboratory confirmation of malaria. However, it is time-consuming and lacks sensitivity while requiring highly skilled laboratory professionals. This study validated a multiplex real-time PCR test for the detection, speciation, and semiquantification of Plasmodium species in whole blood. Analytical results showed the assays were highly specific and sensitive, with a PCR efficiency of 100.21% and 101.99% for Plasmodium falciparum and Plasmodium vivax, respectively. The limit of detection was 0.0001% parasitemia for both P. falciparum and P. vivax with remarkable precision. Accuracy was validated by testing 128 EDTA-whole blood samples from patients suspected of having malaria, with only two discordant results (positive percentage agreement = 98.65%; negative percentage agreement = 98.15%). Moreover, the linearity between cycle threshold value and parasite density (R2 = 0.941) was validated by testing samples serially collected over a 12-day period from a patient positive for P. falciparum, which enabled semiquantitative estimation of parasitemia. This malaria PCR test demonstrates high sensitivity, specificity, reproducibility with minimal hands-on time, and a quick technical turnaround time of <2 hours with capability of providing parasite density estimation to improve diagnosis and monitor treatment of malaria.