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Background  A simple real-time PCR assay using one set of primer and probe for rapid, sensitive and quantitative detection of  Plasmodium  species, with simultaneous differentiation of  Plasmodium falciparum  from the three other  Plasmodium  species ( Plasmodium vivax, Plasmodium ovale  and  Plasmodium malariae ) in febrile returning travellers and migrants was developed and evaluated.    Methods  Consensus primers were used to amplify a species-specific region of the multicopy 18S rRNA gene, and fluorescence resonance energy transfer hybridization probes were used for detection in a LightCycler platform (Roche). The anchor probe sequence was designed to be perfect matches to the 18S rRNA gene of the fourth  Plasmodium  species, while the acceptor probe sequence was designed for  P. falciparum  over a region containing one mismatched, which allowed differentiation of the three other  Plasmodium  species. The performance characteristics of the real-time PCR assay were compared with those of conventional PCR and microscopy-based diagnosis from 119 individuals with a suspected clinical diagnostic of imported malaria.    Results  Blood samples with parasite densities less than 0.01% were all detected, and analytical sensitivity was 0.5 parasite per PCR reaction. The melt curve means Tms (standard deviation) in clinical isolates were 60.5°C (0.6°C) for  P. falciparum  infection and 64.6°C (1.8°C) for non- P. falciparum  species. These Tms values of the  P. falciparum  or non- P. falciparum  species did not vary with the geographic origin of the parasite. The real-time PCR results correlated with conventional PCR using both genus-specific (Kappa coefficient: 0.95, 95% confidence interval: 0.9 – 1) or  P. falciparum -specific (0.91, 0.8 – 1) primers, or with the microscopy results (0.70, 0.6 – 0.8). The real-time assay was 100% sensitive and specific for differentiation of  P. falciparum  to non- P. falciparum  species, compared with conventional PCR or microscopy. The real-time PCR assay can also detect individuals with mixed infections ( P. falciparum  and non- P. falciparum  sp.) in the same sample.    Conclusion  This real-time PCR assay with melting curve analysis is rapid, and specific for the detection and differentiation of  P. falciparum  to other  Plasmodium  species. The suitability for routine use of this assay in clinical diagnostic laboratories is discussed.

Evaluation of FRET real-time PCR assay for rapid detection and differentiation of Plasmodium species in returning travellers and migrants