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

Tran TM, Aghili A, Li S, Ongoiba A, Kayentao K, Doumbo S, Traore B, Crompton PD - Malar. J. (2014)

Bottom Line: Parasite densities determined by nested qPCR, qPCR only, and light microscopy were compared.Nested qPCR results in 10-fold higher sensitivity (0.5 parasites/μl) when compared to qPCR only (five parasites/ul).Nested qPCR improves the sensitivity for the detection of P. falciparum blood-stage infection from clinical DBS samples.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Twinbrook 2, Room 125, 12441 Parklawn Drive, Rockville, Maryland 20852, USA. tuan.tran@nih.gov.

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.

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Related in: MedlinePlus

Correlation between parasite densities determined by light microscopy and calculated by nested qPCR. Calculated parasite densities from nested qPCR were plotted against parasite densities determined by light microscopy for symptomatic infections (red circles), asymptomatic infections with positive blood smears (black circles), and asymptomatic infections with negative blood smears (unfilled circles). Parasite densities calculated by nested PCR strongly correlated with both asymptomatic (Pearson’s r = 0.58, 95% CI [0.29 to 0.77], P < 0.001) and symptomatic (Pearson’s r 0.70, 95% CI [0.53 to 0.81], P < 0.0001) P. falciparum infections. The dashed line represents the limit of detection for blood-smear microscopy (~40 parasites/μl).
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Fig3: Correlation between parasite densities determined by light microscopy and calculated by nested qPCR. Calculated parasite densities from nested qPCR were plotted against parasite densities determined by light microscopy for symptomatic infections (red circles), asymptomatic infections with positive blood smears (black circles), and asymptomatic infections with negative blood smears (unfilled circles). Parasite densities calculated by nested PCR strongly correlated with both asymptomatic (Pearson’s r = 0.58, 95% CI [0.29 to 0.77], P < 0.001) and symptomatic (Pearson’s r 0.70, 95% CI [0.53 to 0.81], P < 0.0001) P. falciparum infections. The dashed line represents the limit of detection for blood-smear microscopy (~40 parasites/μl).

Mentions: Parasite densities derived from nested qPCR were correlated to parasite densities determined by microscopy of concurrent blood smears for smear-positive patients who were either symptomatic or asymptomatic at the time of DBS collection (Figure 3). Parasite densities calculated by nested PCR correlated strongly with both asymptomatic (Pearson’s r = 0.58, 95% CI [0.29 to 0.77], P < 0.001) and symptomatic (Pearson’s r 0.70, 95% CI [0.53 to 0.81], P < 0.0001) P. falciparum infections. For asymptomatic patients who had negative blood smears but were positive by the non-quantitative nested PCR screen, the median parasite density derived from nested qPCR was 220 parasites/μl (interquartile range, 3.6-5100).Figure 3


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

Tran TM, Aghili A, Li S, Ongoiba A, Kayentao K, Doumbo S, Traore B, Crompton PD - Malar. J. (2014)

Correlation between parasite densities determined by light microscopy and calculated by nested qPCR. Calculated parasite densities from nested qPCR were plotted against parasite densities determined by light microscopy for symptomatic infections (red circles), asymptomatic infections with positive blood smears (black circles), and asymptomatic infections with negative blood smears (unfilled circles). Parasite densities calculated by nested PCR strongly correlated with both asymptomatic (Pearson’s r = 0.58, 95% CI [0.29 to 0.77], P < 0.001) and symptomatic (Pearson’s r 0.70, 95% CI [0.53 to 0.81], P < 0.0001) P. falciparum infections. The dashed line represents the limit of detection for blood-smear microscopy (~40 parasites/μl).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4197274&req=5

Fig3: Correlation between parasite densities determined by light microscopy and calculated by nested qPCR. Calculated parasite densities from nested qPCR were plotted against parasite densities determined by light microscopy for symptomatic infections (red circles), asymptomatic infections with positive blood smears (black circles), and asymptomatic infections with negative blood smears (unfilled circles). Parasite densities calculated by nested PCR strongly correlated with both asymptomatic (Pearson’s r = 0.58, 95% CI [0.29 to 0.77], P < 0.001) and symptomatic (Pearson’s r 0.70, 95% CI [0.53 to 0.81], P < 0.0001) P. falciparum infections. The dashed line represents the limit of detection for blood-smear microscopy (~40 parasites/μl).
Mentions: Parasite densities derived from nested qPCR were correlated to parasite densities determined by microscopy of concurrent blood smears for smear-positive patients who were either symptomatic or asymptomatic at the time of DBS collection (Figure 3). Parasite densities calculated by nested PCR correlated strongly with both asymptomatic (Pearson’s r = 0.58, 95% CI [0.29 to 0.77], P < 0.001) and symptomatic (Pearson’s r 0.70, 95% CI [0.53 to 0.81], P < 0.0001) P. falciparum infections. For asymptomatic patients who had negative blood smears but were positive by the non-quantitative nested PCR screen, the median parasite density derived from nested qPCR was 220 parasites/μl (interquartile range, 3.6-5100).Figure 3

Bottom Line: Parasite densities determined by nested qPCR, qPCR only, and light microscopy were compared.Nested qPCR results in 10-fold higher sensitivity (0.5 parasites/μl) when compared to qPCR only (five parasites/ul).Nested qPCR improves the sensitivity for the detection of P. falciparum blood-stage infection from clinical DBS samples.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Twinbrook 2, Room 125, 12441 Parklawn Drive, Rockville, Maryland 20852, USA. tuan.tran@nih.gov.

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.

Show MeSH
Related in: MedlinePlus