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


CONCLUSIONS
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.
Mens
Petra F
PF
Koninklijk Instituut voor de Tropen/Royal Tropical Institute, KIT Biomedical Research, Meibergdreef 39, 1105 AZ Amsterdam, The Netherlands. p.mens@kit.nl
Schoone
Gerard J
GJ
Kager
Piet A
PA
Schallig
Henk D F H
HD
D000818:Animals; D001483:Base Sequence; D003199:Computer Systems; D016054:DNA, Protozoan / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D006801:Humans; D008288:Malaria / Q000175:diagnosis / Q000469:parasitology*; D008969:Molecular Sequence Data; D018512:Parasitemia / Q000469:parasitology; D010961:Plasmodium / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D010963:Plasmodium falciparum / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D010965:Plasmodium malariae / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D041122:Plasmodium ovale / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D010966:Plasmodium vivax / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D016053:RNA, Protozoan / Q000235:genetics; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D021161:Self-Sustained Sequence Replication / Q000379:methods*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D016037:Single-Blind Method; D013045:Species Specificity
D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D016053:RNA, Protozoan; D012337:RNA, Ribosomal, 18S