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Detection and identification of human Plasmodium species with real-time quantitative nucleic acid sequence-based amplification.
Malar J. 2006 Oct 03; 5:80.MJ

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.

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Authors+Show Affiliations

Mens PF
Koninklijk Instituut voor de Tropen/Royal Tropical Institute, KIT Biomedical Research, Meibergdreef 39, 1105 AZ Amsterdam, The Netherlands. p.mens@kit.nl
Schoone GJ
No affiliation info available
Kager PA
No affiliation info available
Schallig HD
No affiliation info available

MeSH

AnimalsBase SequenceComputer SystemsDNA, ProtozoanDNA, RibosomalHumansMalariaMolecular Sequence DataParasitemiaPlasmodiumPlasmodium falciparumPlasmodium malariaePlasmodium ovalePlasmodium vivaxRNA, ProtozoanRNA, Ribosomal, 18SSelf-Sustained Sequence ReplicationSensitivity and SpecificitySequence AlignmentSequence Homology, Nucleic AcidSingle-Blind MethodSpecies Specificity

Pub Type(s)

Comparative Study
Evaluation Study
Journal Article
Research Support, Non-U.S. Gov't
Validation Study

Language

eng

PubMed ID

17018138

Citation

Mens, Petra F., et al. "Detection and Identification of Human Plasmodium Species With Real-time Quantitative Nucleic Acid Sequence-based Amplification." Malaria Journal, vol. 5, 2006, p. 80.
Mens PF, Schoone GJ, Kager PA, et al. Detection and identification of human Plasmodium species with real-time quantitative nucleic acid sequence-based amplification. Malar J. 2006;5:80.
Mens, P. F., Schoone, G. J., Kager, P. A., & Schallig, H. D. (2006). Detection and identification of human Plasmodium species with real-time quantitative nucleic acid sequence-based amplification. Malaria Journal, 5, 80.
Mens PF, et al. Detection and Identification of Human Plasmodium Species With Real-time Quantitative Nucleic Acid Sequence-based Amplification. Malar J. 2006 Oct 3;5:80. PubMed PMID: 17018138.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Detection and identification of human Plasmodium species with real-time quantitative nucleic acid sequence-based amplification. AU - Mens,Petra F, AU - Schoone,Gerard J, AU - Kager,Piet A, AU - Schallig,Henk D F H, Y1 - 2006/10/03/ PY - 2006/09/04/received PY - 2006/10/03/accepted PY - 2006/10/5/pubmed PY - 2006/11/10/medline PY - 2006/10/5/entrez SP - 80 EP - 80 JF - Malaria journal JO - Malar J VL - 5 N2 - 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. SN - 1475-2875 UR - https://www.unboundmedicine.com/medline/citation/17018138/Detection_and_identification_of_human_Plasmodium_species_with_real_time_quantitative_nucleic_acid_sequence_based_amplification_ L2 - https://malariajournal.biomedcentral.com/articles/10.1186/1475-2875-5-80 DB - PRIME DP - Unbound Medicine ER -