Background

Microscopy of Giemsa stained blood smears is still the gold standard for malaria diagnostics (Yin et al., 2018). However, microscopy requires considerable expertise, particularly at low-level parasitemia, which is often seen in imported malaria in non-endemic countries. Moreover, differentiating the Plasmodium species based on their morphological characteristics can be demanding, especially after chemoprophylaxis or auto-medication (Calderaro et al., 2018). Molecular detection and identification of Plasmodium spp. is a highly accurate and sensitive alternative method for the diagnosis of malaria. Particularly, real-time PCR has the advantage of fast and quantitative results and, compared to nested PCR, the risk of contamination is reduced. However, many published Plasmodium real-time PCR protocols have limitations, e.g., do not differentiate between species (Safeukui et al., 2008; Haanshuus et al., 2019; Farcas et al., 2004), do not detect all species (Perandin et al., 2004;Kim et al., 2014; Veron et al., 2009), or a positive genus-specific PCR has to be followed by two additional multiplex reactions, to obtain a species-specific result (Rougemont et al., 2004).

Hence, we developed and evaluated a new fluorescence resonance energy transfer-based real-time PCR (FRET-qPCR) that allows a quantitative, rapid, sensitive, and species-specific diagnosis of malaria (Schneider et al., 2021). The sequences of the hybridization probes MalFL and MalLC640 match P. falciparum. Fluorescence at 640 nm was generated by FRET, following the annealing of both probes to their adjacent complementary sequences. Species discrimination is based on the presence of single nucleotide polymorphisms (SNPs), reducing the affinity of the MalFLprobe for P. vivax/knowlesi (2 mismatches), P. ovale (1 mismatch), and P. malariae (1 mismatch), and thus lowering the melting temperature (Tm) in the melting curve analysis (Figure 1). The current paper is a step-by-step protocol for the performance of this new FRET-qPCR, focusing on the proper handling and storage of all components. The details described in this protocol are crucial comments to achieve excellent amplification and melting curves, a prerequisite for correct species identification based on single nucleotide polymorphisms (SNPs).

The advantage of requiring only one reaction per patient, and the short turn-around time of less than two hours (including DNA extraction), makes it a valuable diagnostic tool, especially in the absence of experienced microscopists. Moreover, results are objective and reproducible, DNA extraction can be automated, and the use of only one set of primers and probes is a significant advantage in routine clinical applications. One technician, well-trained in molecular methods, will be able to test a large number of patient samples in a relatively short turn-around time.

This protocol can be helpful for the rapid and reliable diagnosis of malaria in individual patients, particularly travelers returning from endemic areas, migrants, and refugees. Another field of application is treatment monitoring, by evaluating the quantitative results, and the crossing point (Cp) values. These allow the determination of the reduction in parasite number, important to detect early treatment failure (Rougemont et al., 2004). The potential of the FRET-qPCRs to detect asymptomatic infections can be useful for screening travelers returning to non-endemic regions. Moreover, it can also be performed in well-equipped malaria reference laboratories in endemic countries.