Sensitivity Assessment of a Multiplex and Real-Time PCR Protocols for the Detection of Malaria in External Quality Control Samples in the Malaria Reference Center in Greece

Abstract

Background: Accurate malaria diagnosis constitutes a challenging task, necessitating the need for the implementation of targeted and effective diagnostic tools. The purpose of the current study was to evaluate the effectiveness of two different molecular methodologies in terms of sensitivity for the detection of External Quality Assessment (EQA) Plasmodium samples. Methods: A total of 104 lyophilized blood samples from 14 different UK-NEQAS (National External Quality Assessment Site) (2016–2021) and eight WHO-NEQAS distributions (2017–2020) were analyzed. An in-house multiplex PCR protocol, followed by single target real-time PCR protocols for all five Plasmodium species, was implemented. Results: The multiplex PCR had a success rate of 10/16 and 20/28 for P. vivax and P. falciparum species, respectively. On the other hand, the respective results for real-time PCR had a success rate of 13/16 (P. vivax), 28/28 (P. falciparum), 5/8 (P. malariae), 8/10 (P. ovale), and 10/14 (P. knowlesi). Plasmodium falciparum samples displayed the highest sensitivity of detection, 0.02 parasites/μL. Plasmodium vivax samples displayed a 0.1 parasites/μL cutoff value, greater than the respective value for whole blood samples, while P. ovale species displayed a respective cutoff value of 0.05 parasites/μL. Due to the limited number of tested samples, data obtained for P. malariae and P. knowlesi species samples were inconclusive. Conclusions: Real-time PCR comprises a credible molecular methodology in terms of sensitivity assessment and detection of low parasitemia levels of Plasmodium sp. in EQA lyophilized blood samples.

Keywords

• malaria
• <i>Plasmodium</i> species
• diagnosis
• multiplex PCR
• real-time PCR