A nested real-time PCR assay for the quantification of Plasmodium falciparum DNA extracted from dried blood spots

Malaria Journal

Methodology

Tuan M Tran¹  Peter D Crompton¹  Amirali Aghili¹  Shanping Li¹  Aissata Ongoiba²  Kassoum Kayentao²  Safiatou Doumbo²  Boubacar Traore² 

[1]Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Twinbrook 2, Room 125, 12441 Parklawn Drive, 20852, Rockville, Maryland, USA

[2]Mali International Center of Excellence in Research, University of Sciences, Techniques, and Technology of Bamako (USTTB), BP: 1805, Point G, Bamako, Mali

关键词: Plasmodium falciparum;    Nucleic acid testing;    Quantitative PCR;    Nested PCR;    Dried blood spot;    Passive surveillance;   

DOI  :  10.1186/1475-2875-13-393

 received in 2014-06-13, accepted in 2014-09-30,  发布年份 2014

来源: Springer

【 摘 要 】

BackgroundAs public health efforts seek to eradicate malaria, there has been an emphasis on eliminating low-density parasite reservoirs in asymptomatic carriers. As such, diagnosing submicroscopic Plasmodium infections using PCR-based techniques has become important not only in clinical trials of malaria vaccines and therapeutics, but also in active malaria surveillance campaigns. However, PCR-based quantitative assays that rely on nucleic acid extracted from dried blood spots (DBS) have demonstrated lower sensitivity than assays that use cryopreserved whole blood as source material.MethodsThe density of Plasmodium falciparum asexual parasites was quantified using genomic DNA extracted from dried blood spots (DBS) and the sensitivity of two approaches was compared: quantitative real-time PCR (qPCR) targeting the P. falciparum 18S ribosomal RNA gene, either with an initial conventional PCR amplification prior to qPCR (nested qPCR), or without an initial amplification (qPCR only). Parasite densities determined by nested qPCR, qPCR only, and light microscopy were compared.ResultsNested qPCR results in 10-fold higher sensitivity (0.5 parasites/μl) when compared to qPCR only (five parasites/ul). Among microscopy-positive samples, parasite densities calculated by nested qPCR correlated strongly with microscopy for both asymptomatic (Pearson’s r = 0.58, P < 0.001) and symptomatic (Pearson’s r = 0.70, P < 0.0001) P. falciparum infections.ConclusionNested qPCR improves the sensitivity for the detection of P. falciparum blood-stage infection from clinical DBS samples. This approach may be useful for active malaria surveillance in areas where submicroscopic asymptomatic infections are prevalent.

【 授权许可】

CC BY   
© Tran et al.; licensee BioMed Central Ltd. 2014