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. We validated a multiplex real-time PCR test for the detection, speciation, and semi-quantification 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 P. falciparum and P. 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 malaria with only 2 discordant results (PPA=98.65%; NPA=98.15%). Moreover, we validated the linearity between Ct value and parasite density (R 2 = 0.941) by testing samples serially collected over a 12-day period from a patient positive for P. falciparum , which enabled semi-quantitative 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.