Implementation of a Composite Reference Standard Methodology in the Greek National Malaria Reference Center, Insights from a Seven-Year Experience

Background: Malaria is currently ranked as an important public health issue in Greece despite its initial eradication back in 1974. Given that the clinical picture is often nonspecific, the need for the implementation of targeted laboratory methodologies is imperative to ensure prompt Plasmodium identification and diagnosis. Methods: A total of 780 clinical samples from 763 suspected malaria cases from the National Malaria Reference Center were analyzed within a seven-year period (2017–2023). A composite laboratory reference standard methodology, including rapid diagnostic testing, microscopy, and polymerase chain reactions, was implemented for the purposes set by the current study in a group of 532 samples. A multivariable statistical analysis was implemented for the interpretation of the extracted data. Results: Microscopy and PCR successfully identified P. falciparum (111 vs. 131) and P. vivax (95 vs. 95) clinical samples. In addition, microscopy successfully identified selected cases of P. ovale (16) and P. malariae (nine), with current cases being confirmed by targeted, species-level real-time PCR protocols. Conclusions: The current study highlighted the importance of microscopic examination in malaria diagnostic procedures for Plasmodium species identification and typing. The combination of microscopy and PCR ensures a credible laboratory approach for submicroscopic clinical cases and patients with prior antimalarial treatment. This composite laboratory reference standard methodology is recommended by the Greek Malaria Reference Center for prompt identification of Plasmodium species in suspected malaria clinical cases.