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Combined DNA extraction and antibody elution from filter papers for the assessment of malaria transmission intensity in epidemiological studies.

Baidjoe A, Stone W, Ploemen I, Shagari S, Grignard L, Osoti V, Makori E, Stevenson J, Kariuki S, Sutherland C, Sauerwein R, Cox J, Drakeley C, Bousema T - Malar. J. (2013)

Bottom Line: The eluate of the first wash during the DNA extraction process was used for antibody detection and compared with previously validated antibody elution procedures.Estimates of antibody prevalence are unaffected by the combined extraction and elution procedure.The choice of target gene and the amount and source of filter paper material for DNA extraction can have a marked impact on PCR sensitivity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands.

ABSTRACT

Background: Informing and evaluating malaria control efforts relies on knowledge of local transmission dynamics. Serological and molecular tools have demonstrated great sensitivity to quantify transmission intensity in low endemic settings where the sensitivity of traditional methods is limited. Filter paper blood spots are commonly used a source of both DNA and antibodies. To enhance the operational practicability of malaria surveys, a method is presented for combined DNA extraction and antibody elution.

Methods: Filter paper blood spots were collected as part of a large cross-sectional survey in the Kenyan highlands. DNA was extracted using a saponin/chelex method. The eluate of the first wash during the DNA extraction process was used for antibody detection and compared with previously validated antibody elution procedures. Antibody elution efficiency was assessed by total IgG ELISA for malaria antigens apical membrane antigen-1 (AMA-1) and merozoite-surface protein-1 (MSP-142). The sensitivity of nested 18S rRNA and cytochrome b PCR assays and the impact of doubling filter paper material for PCR sensitivity were determined. The distribution of cell material and antibodies throughout filter paper blood spots were examined using luminescent and fluorescent reporter assays.

Results: Antibody levels measured after the combined antibody/DNA extraction technique were strongly correlated to those measured after standard antibody elution (p < 0.0001). Antibody levels for both AMA-1 and MSP-142 were generally slightly lower (11.3-21.4%) but age-seroprevalence patterns were indistinguishable. The proportion of parasite positive samples ranged from 12.9% to 19.2% in the different PCR assays. Despite strong agreement between outcomes of different PCR assays, none of the assays detected all parasite-positive individuals. For all assays doubling filter paper material for DNA extraction increased sensitivity. The concentration of cell and antibody material was not homogenously distributed throughout blood spots.

Conclusion: Combined DNA extraction and antibody elution is an operationally attractive approach for high throughput assessment of cumulative malaria exposure and current infection prevalence in endemic settings. Estimates of antibody prevalence are unaffected by the combined extraction and elution procedure. The choice of target gene and the amount and source of filter paper material for DNA extraction can have a marked impact on PCR sensitivity.

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Related in: MedlinePlus

DNA/Cell and antibody distribution on filter paper. A. Photograph of one of the two blood spots analysed for cell material distribution, in which luminescence intensity is proportional to the density of cell material. Luminescence intensity values in the cells of the overlying grid are calculated as a proportion of the highest cell value, and are presented in the adjacent grid schema. B. Photograph of one of the two blood spots analysed for antibody material distribution, in which fluorescence intensity is proportional to the density of antibodies. Fluorescence intensity values are calculated and presented as in Figure 3A.
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Figure 3: DNA/Cell and antibody distribution on filter paper. A. Photograph of one of the two blood spots analysed for cell material distribution, in which luminescence intensity is proportional to the density of cell material. Luminescence intensity values in the cells of the overlying grid are calculated as a proportion of the highest cell value, and are presented in the adjacent grid schema. B. Photograph of one of the two blood spots analysed for antibody material distribution, in which fluorescence intensity is proportional to the density of antibodies. Fluorescence intensity values are calculated and presented as in Figure 3A.

Mentions: Luminescence produced by GFP expression in Pb-GFPluccon infected blood samples was previously shown to correlate strongly with parasitaemia [39]. Here, the distribution of DNA on filter paper was assessed by measuring the luminescent intensity in dried blood spots from mice infected with Pb-GFPluccon[38]. Cell by cell luminescence analysis of the grid overlaying the blood spot was used to describe heterogeneity in parasite material in different parts of the blood spot. Both blood spots tested showed a considerable degree of heterogeneity in the distribution of parasite material (Figure 3). In the two separate experiments, 25% and 64% of the grid cells contained less than 85% of the parasite material of the grid cell with the highest quantity. Parasite material seemed less concentrated towards the extreme edges of the blood spot; the grid cell with the lowest parasite quantity contained 70% of the maximum value (Figure 3).


Combined DNA extraction and antibody elution from filter papers for the assessment of malaria transmission intensity in epidemiological studies.

Baidjoe A, Stone W, Ploemen I, Shagari S, Grignard L, Osoti V, Makori E, Stevenson J, Kariuki S, Sutherland C, Sauerwein R, Cox J, Drakeley C, Bousema T - Malar. J. (2013)

DNA/Cell and antibody distribution on filter paper. A. Photograph of one of the two blood spots analysed for cell material distribution, in which luminescence intensity is proportional to the density of cell material. Luminescence intensity values in the cells of the overlying grid are calculated as a proportion of the highest cell value, and are presented in the adjacent grid schema. B. Photograph of one of the two blood spots analysed for antibody material distribution, in which fluorescence intensity is proportional to the density of antibodies. Fluorescence intensity values are calculated and presented as in Figure 3A.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750228&req=5

Figure 3: DNA/Cell and antibody distribution on filter paper. A. Photograph of one of the two blood spots analysed for cell material distribution, in which luminescence intensity is proportional to the density of cell material. Luminescence intensity values in the cells of the overlying grid are calculated as a proportion of the highest cell value, and are presented in the adjacent grid schema. B. Photograph of one of the two blood spots analysed for antibody material distribution, in which fluorescence intensity is proportional to the density of antibodies. Fluorescence intensity values are calculated and presented as in Figure 3A.
Mentions: Luminescence produced by GFP expression in Pb-GFPluccon infected blood samples was previously shown to correlate strongly with parasitaemia [39]. Here, the distribution of DNA on filter paper was assessed by measuring the luminescent intensity in dried blood spots from mice infected with Pb-GFPluccon[38]. Cell by cell luminescence analysis of the grid overlaying the blood spot was used to describe heterogeneity in parasite material in different parts of the blood spot. Both blood spots tested showed a considerable degree of heterogeneity in the distribution of parasite material (Figure 3). In the two separate experiments, 25% and 64% of the grid cells contained less than 85% of the parasite material of the grid cell with the highest quantity. Parasite material seemed less concentrated towards the extreme edges of the blood spot; the grid cell with the lowest parasite quantity contained 70% of the maximum value (Figure 3).

Bottom Line: The eluate of the first wash during the DNA extraction process was used for antibody detection and compared with previously validated antibody elution procedures.Estimates of antibody prevalence are unaffected by the combined extraction and elution procedure.The choice of target gene and the amount and source of filter paper material for DNA extraction can have a marked impact on PCR sensitivity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands.

ABSTRACT

Background: Informing and evaluating malaria control efforts relies on knowledge of local transmission dynamics. Serological and molecular tools have demonstrated great sensitivity to quantify transmission intensity in low endemic settings where the sensitivity of traditional methods is limited. Filter paper blood spots are commonly used a source of both DNA and antibodies. To enhance the operational practicability of malaria surveys, a method is presented for combined DNA extraction and antibody elution.

Methods: Filter paper blood spots were collected as part of a large cross-sectional survey in the Kenyan highlands. DNA was extracted using a saponin/chelex method. The eluate of the first wash during the DNA extraction process was used for antibody detection and compared with previously validated antibody elution procedures. Antibody elution efficiency was assessed by total IgG ELISA for malaria antigens apical membrane antigen-1 (AMA-1) and merozoite-surface protein-1 (MSP-142). The sensitivity of nested 18S rRNA and cytochrome b PCR assays and the impact of doubling filter paper material for PCR sensitivity were determined. The distribution of cell material and antibodies throughout filter paper blood spots were examined using luminescent and fluorescent reporter assays.

Results: Antibody levels measured after the combined antibody/DNA extraction technique were strongly correlated to those measured after standard antibody elution (p < 0.0001). Antibody levels for both AMA-1 and MSP-142 were generally slightly lower (11.3-21.4%) but age-seroprevalence patterns were indistinguishable. The proportion of parasite positive samples ranged from 12.9% to 19.2% in the different PCR assays. Despite strong agreement between outcomes of different PCR assays, none of the assays detected all parasite-positive individuals. For all assays doubling filter paper material for DNA extraction increased sensitivity. The concentration of cell and antibody material was not homogenously distributed throughout blood spots.

Conclusion: Combined DNA extraction and antibody elution is an operationally attractive approach for high throughput assessment of cumulative malaria exposure and current infection prevalence in endemic settings. Estimates of antibody prevalence are unaffected by the combined extraction and elution procedure. The choice of target gene and the amount and source of filter paper material for DNA extraction can have a marked impact on PCR sensitivity.

Show MeSH
Related in: MedlinePlus