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A Field-Tailored Reverse Transcription Loop-Mediated Isothermal Assay for High Sensitivity Detection of Plasmodium falciparum Infections.
- Source :
- PLoS ONE, Vol 11, Iss 11, p e0165506 (2016)
- Publication Year :
- 2016
- Publisher :
- Public Library of Science (PLoS), 2016.
-
Abstract
- Highly sensitive and field deployable molecular diagnostic tools are critically needed for detecting submicroscopic, yet transmissible levels of malaria parasites prevalent in malaria endemic countries worldwide. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed and evaluated in comparison with thick blood smear microscopy, an antigen-based rapid diagnostic test (RDT), and an in-house RT-PCR targeting the same RT-LAMP transcript. The optimized assay detected Plasmodium falciparum infections in as little as 0.25ng of total parasite RNA, and exhibited a detection limit of 0.08 parasites/ μL when tested directly on infected whole blood lysates, or ~0.0008 parasites/ μL when using RNA extracts. Assay positivity was observed as early as eight minutes from initiation of the RT-LAMP and in most cases the reaction was complete before twenty minutes. Clinical evaluation of the assay on 132 suspected malaria cases resulted in a positivity rate of 90% for RT-LAMP using extracted RNA, and 85% when using whole blood lysates. The positivity rates were 70% for P. falciparum-specific RDT, 83% for RT-PCR, and 74% for thick blood smear microscopy (Mean parasite density = 36,986 parasites/ μL). Concordance rates between the developed RT-LAMP and comparator tests were greater than 75%, the lowest being with light microscopy (78%, McNemar's test: P = 0.0002), and the highest was with RT-PCR (87%, McNemar's test: P = 0.0523). Compared to reference RT-PCR, assay sensitivity was 90% for RT-LAMP on whole blood, and 96% for RT-LAMP using corresponding RNA extracts. Electricity-free heaters were further developed and evaluated in comparison with a battery-operated isothermal amplification machine for use with the developed test in resource-limited settings. Taken together, the data highlight the benefits of targeting high abundant RNA transcripts in molecular diagnosis, as well as the potential usefulness of the developed RT-LAMP-assay in malaria diagnosis in low to high parasite density settings.
Details
- Language :
- English
- ISSN :
- 19326203
- Volume :
- 11
- Issue :
- 11
- Database :
- Directory of Open Access Journals
- Journal :
- PLoS ONE
- Publication Type :
- Academic Journal
- Accession number :
- edsdoj.9c8bc10a997043a3a5a0bf8480cbfd2d
- Document Type :
- article
- Full Text :
- https://doi.org/10.1371/journal.pone.0165506