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

Tuan  Tran, Amirali Aghili, Shanping Li, Aissata Ongoiba, Kassoum Kayentao, Safiatou Doumbo, Boubacar Traore, Peter D. Crompton

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

Background: As 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.

Methods. The 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.

Results: Nested 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.

Conclusion: Nested 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.

Original languageEnglish (US)
Article number393
JournalMalaria Journal
Volume13
Issue number1
DOIs
StatePublished - Oct 4 2014
Externally publishedYes

Fingerprint

Plasmodium falciparum
Real-Time Polymerase Chain Reaction
Parasites
Malaria
DNA
Microscopy
Polymerase Chain Reaction
18S Ribosomal RNA
Malaria Vaccines
Asymptomatic Infections
rRNA Genes
Nucleic Acids
Public Health
Clinical Trials
Light
Infection

Keywords

  • Dried blood spot
  • Nested PCR
  • Nucleic acid testing
  • Passive surveillance
  • Plasmodium falciparum
  • Quantitative PCR

ASJC Scopus subject areas

  • Infectious Diseases
  • Parasitology
  • Medicine(all)

Cite this

A nested real-time PCR assay for the quantification of Plasmodium falciparum DNA extracted from dried blood spots. / Tran, Tuan ; Aghili, Amirali; Li, Shanping; Ongoiba, Aissata; Kayentao, Kassoum; Doumbo, Safiatou; Traore, Boubacar; Crompton, Peter D.

In: Malaria Journal, Vol. 13, No. 1, 393, 04.10.2014.

Research output: Contribution to journalArticle

Tran, Tuan  ; Aghili, Amirali ; Li, Shanping ; Ongoiba, Aissata ; Kayentao, Kassoum ; Doumbo, Safiatou ; Traore, Boubacar ; Crompton, Peter D. / A nested real-time PCR assay for the quantification of Plasmodium falciparum DNA extracted from dried blood spots. In: Malaria Journal. 2014 ; Vol. 13, No. 1.
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