Detection and identification of human Plasmodium species with real-time quantitative nucleic acid sequence-based amplification

Abstract

Background: Decisions concerning malaria treatment depend on species identification causing disease. Microscopy is most frequently used, but at low parasitaemia (<20 parasites/mul) the technique becomes less sensitive and time consuming. Rapid diagnostic tests based on Plasmodium antigen detection do often not allow for species discrimination as microscopy does, but also become insensitive at <100 parasites/microl.

Methods: This paper reports the development of a sensitive and specific real-time Quantitative Nucleic Acid Sequence Based Amplification (real-time QT-NASBA) assays, based on the small-subunit 18S rRNA gene, to identify the four human Plasmodium species.

Results: The lower detection limit of the assay is 100-1000 molecules in vitro RNA for all species, which corresponds to 0.01-0.1 parasite per diagnostic sample (i.e. 50 microl of processed blood). The real-time QT-NASBA was further evaluated using 79 clinical samples from malaria patients: i.e. 11 Plasmodium. falciparum, 37 Plasmodium vivax, seven Plasmodium malariae, four Plasmodium ovale and 20 mixed infections. The initial diagnosis of 69 out of the 79 samples was confirmed with the developed real-time QT-NASBA. Re-analysis of seven available original slides resolved five mismatches. Three of those were initially identified as P. malariae mono-infection, but after re-reading the slides P. falciparum was found, confirming the real-time QT-NASBA result. The other two slides were of poor quality not allowing true species identification. The remaining five discordant results could not be explained by microscopy, but may be due to extreme low numbers of parasites present in the samples. In addition, 12 Plasmodium berghei isolates from mice and 20 blood samples from healthy donors did not show any reaction in the assay.

Conclusion: Real-time QT-NASBA is a very sensitive and specific technique with a detection limit of 0.1 Plasmodium parasite per diagnostic sample (50 microl of blood) and can be used for the detection, identification and quantitative measurement of low parasitaemia of Plasmodium species, thus making it an effective tool for diagnostic purposes and useful for epidemiological and drug studies.

Figure 1

Alignment of sequenced 18SrRNA gene. Forward primers used in the different assays are shown in italics. Beacon and reverse primers are shown in bold.

Figure 2

Standard curves of the P. malariae assay. A standard curve of the NASBA assay with P. malariae in vitro RNA using the P. malariae specific forward and generic revere primer and beacon. R² = 0,972.

Figure 3

Standard curves of the P. vivax assay. A standard curve of the NASBA assay with P. vivax in vitro RNA using the P. vivax specific forward and generic revere primer and beacon wit an R² = 0,992.

Figure 4

Standard curves of the P. ovale assay and figure c the standard curve of the NASBA assay with P. ovale in vitro RNA using the P. ovale specific forward and generic revere primer and beacon. R² = 0,993.