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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

Comparison of quantitative real-time PCR standard curves. 10-fold plasmid or gDNA dilutions were plotted against Ct values generated from qPCR only and nested qPCR assays using (A)P. falciparum 18S rRNA plasmid or (B) gDNA extracted from dried blood spots obtained from a subject with clinical malaria as template DNA (described in the Methods). Points represent the mean of technical duplicates and error bars (where visible) indicate the standard deviation. Dotted lines represent the technical limit of detection as defined as the lowest template concentration for which there is a linear relationship between Ct values and copy number/parasite density. For (C), Ct values generated by nested qPCR were plotted against the parasite densities estimated from copy number for P. falciparum 18S rRNA plasmid or corresponding to the dilutions from the clinical DBS standards. The best-fit regression line is shown as a black line.
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Fig1: Comparison of quantitative real-time PCR standard curves. 10-fold plasmid or gDNA dilutions were plotted against Ct values generated from qPCR only and nested qPCR assays using (A)P. falciparum 18S rRNA plasmid or (B) gDNA extracted from dried blood spots obtained from a subject with clinical malaria as template DNA (described in the Methods). Points represent the mean of technical duplicates and error bars (where visible) indicate the standard deviation. Dotted lines represent the technical limit of detection as defined as the lowest template concentration for which there is a linear relationship between Ct values and copy number/parasite density. For (C), Ct values generated by nested qPCR were plotted against the parasite densities estimated from copy number for P. falciparum 18S rRNA plasmid or corresponding to the dilutions from the clinical DBS standards. The best-fit regression line is shown as a black line.

Mentions: Standard curves plotting Ct values against starting copy numbers or parasite densities (parasites/μl) were generated from serially diluted plasmid DNA containing the P. falciparum 18S rRNA gene or infected standard, respectively, for both 1) 15-cycle standard PCR amplification followed by nested qPCR and 2) amplification by qPCR only (Figure 1). Based on an estimate of 6 copies of 18S rRNA per P. falciparum genome [10], the limit of detection using P. falciparum 18S rRNA plasmid is 0.17 parasites/μl (equivalent to 1 copy/μl) for nested qPCR and 17 parasites/μl copies by qPCR only (equivalent to 100 copies/μl; Figures 1A and 2A). Similarly, the limit of detection using infected standards is 0.05 parasites/μl for nested qPCR and five parasites/μl for qPCR only (Figures 1B and 2B). To evaluate whether the two different sources of template DNA (P. falciparum 18S rRNA plasmid versus infected standard) yielded directly comparable parasite density estimates based on their Ct values, the Ct values generated by nested qPCR were plotted against the parasite densities estimated from copy number for P. falciparum 18S rRNA plasmid or corresponding to the dilutions in the case of infected standards (Figure 1C). A strong, negative correlation existed between parasite density and Ct values when values from both sources were combined (Pearson’s r −0.99; 95% CI [−1.0 to −0.96]; P < 0.001). High Ct values (>33 cycles) generated in samples with no template during the first amplification (Figure 2A) were confirmed as negative by inspection of dissociation curves (Additional file 1).Figure 1


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)

Comparison of quantitative real-time PCR standard curves. 10-fold plasmid or gDNA dilutions were plotted against Ct values generated from qPCR only and nested qPCR assays using (A)P. falciparum 18S rRNA plasmid or (B) gDNA extracted from dried blood spots obtained from a subject with clinical malaria as template DNA (described in the Methods). Points represent the mean of technical duplicates and error bars (where visible) indicate the standard deviation. Dotted lines represent the technical limit of detection as defined as the lowest template concentration for which there is a linear relationship between Ct values and copy number/parasite density. For (C), Ct values generated by nested qPCR were plotted against the parasite densities estimated from copy number for P. falciparum 18S rRNA plasmid or corresponding to the dilutions from the clinical DBS standards. The best-fit regression line is shown as a black line.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Comparison of quantitative real-time PCR standard curves. 10-fold plasmid or gDNA dilutions were plotted against Ct values generated from qPCR only and nested qPCR assays using (A)P. falciparum 18S rRNA plasmid or (B) gDNA extracted from dried blood spots obtained from a subject with clinical malaria as template DNA (described in the Methods). Points represent the mean of technical duplicates and error bars (where visible) indicate the standard deviation. Dotted lines represent the technical limit of detection as defined as the lowest template concentration for which there is a linear relationship between Ct values and copy number/parasite density. For (C), Ct values generated by nested qPCR were plotted against the parasite densities estimated from copy number for P. falciparum 18S rRNA plasmid or corresponding to the dilutions from the clinical DBS standards. The best-fit regression line is shown as a black line.
Mentions: Standard curves plotting Ct values against starting copy numbers or parasite densities (parasites/μl) were generated from serially diluted plasmid DNA containing the P. falciparum 18S rRNA gene or infected standard, respectively, for both 1) 15-cycle standard PCR amplification followed by nested qPCR and 2) amplification by qPCR only (Figure 1). Based on an estimate of 6 copies of 18S rRNA per P. falciparum genome [10], the limit of detection using P. falciparum 18S rRNA plasmid is 0.17 parasites/μl (equivalent to 1 copy/μl) for nested qPCR and 17 parasites/μl copies by qPCR only (equivalent to 100 copies/μl; Figures 1A and 2A). Similarly, the limit of detection using infected standards is 0.05 parasites/μl for nested qPCR and five parasites/μl for qPCR only (Figures 1B and 2B). To evaluate whether the two different sources of template DNA (P. falciparum 18S rRNA plasmid versus infected standard) yielded directly comparable parasite density estimates based on their Ct values, the Ct values generated by nested qPCR were plotted against the parasite densities estimated from copy number for P. falciparum 18S rRNA plasmid or corresponding to the dilutions in the case of infected standards (Figure 1C). A strong, negative correlation existed between parasite density and Ct values when values from both sources were combined (Pearson’s r −0.99; 95% CI [−1.0 to −0.96]; P < 0.001). High Ct values (>33 cycles) generated in samples with no template during the first amplification (Figure 2A) were confirmed as negative by inspection of dissociation curves (Additional file 1).Figure 1

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