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

Abstract

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 degrees C (0.6 degrees C) for P. falciparum infection and 64.6 degrees C (1.8 degrees 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.

Figure 1

Primer and fluorescence probe positions selected for FRET PCR of Plasmodium 18S rRNA gene. Sequences of forward (underline left panel) and reverse (underline right panel) primers were aligned with the corresponding target sequences. FRET probes for detection of parasite (blue and red for anchor and acceptor FRET hybridization probes, respectively). Acceptor FRET hybridization probe was designed on the basis of one nucleotide mismatch difference (shown in pink colour) that distinguish 18S rRNA gene of P. falciparum (GenBank Accession number: AL010278) from that of the P. vivax (GenBank Accession number: U83877) or P. ovale (GenBank Accession number: L48986) or P. malariae (GenBank Accession number: M54897).

Figure 2

Melting curves of amplicons post real-time PCR of DNA extracted from three different blood samples with known P. falciparum, non-P. falciparum (P. ovale) and mixed Plasmodium sp. (P. falciparum + P. ovale). The Tms of the P. falciparum were distinctively lower than that of P. ovale. Negative control included reaction mixture with water.

Figure 3

Sensitivity, inter- and intra-assay variabilities of P. falciparum DNA quantification. (A) Results obtained from four independent tests in duplicate by real-time PCR assay using FRET. (B) Linearity of FRET assay PCR is shown using serially diluted P. falciparum DNA (5000 to 0.25 parasites per reaction, one representative experiment). Amplification efficiency (AE) is calculated based on the slope of the standard curves using the formula: E = 10^1/-s-1, where E(100) is the % efficiency and s is the slope of the standard cure. (C) Amplification of serially diluted P. falciparum DNA (5000 to 5 parasites per PCR reaction) by conventional PCR assay followed by gel electrophoresis and ethidium bromide staining.

Figure 4

Amplicon melting temperature plots (Tms) of P. falciparum and non-P. falciparum sp. according to the geographic origin of infection. Tms of the P. falciparum or Non-P. falciparum species were similar between origin of parasite. n, effective.