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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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Antibody level from standard and dual filter paper blood spot elution methods for AMA-1 and MSP-142. A. Scatter plot showing anti-AMA-1 IgG level detected in 236 individuals by standard (x-axis) and combined (y-axis) elution of filter paper blood spots. R2 (linear regression) = 0.93 (p = <0.0001). B. Scatter plot showing anti-MSP-142 IgG level detected in 236 individuals using standard (x-axis) and combined (y-axis) elution of filter paper blood spots. R2 (linear regression) 0.671 (p = <0.0001).
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Figure 1: Antibody level from standard and dual filter paper blood spot elution methods for AMA-1 and MSP-142. A. Scatter plot showing anti-AMA-1 IgG level detected in 236 individuals by standard (x-axis) and combined (y-axis) elution of filter paper blood spots. R2 (linear regression) = 0.93 (p = <0.0001). B. Scatter plot showing anti-MSP-142 IgG level detected in 236 individuals using standard (x-axis) and combined (y-axis) elution of filter paper blood spots. R2 (linear regression) 0.671 (p = <0.0001).

Mentions: Antibodies were eluted from 236 filter papers by both the standard elution procedure and the combined elution procedure. For both AMA-1 and MSP-142, a strong positive correlation was observed between the IgG responses of filter paper blood samples eluted by standard and combined methods (Figure 1). While strongly correlated, there was a tendency toward higher antibody responses when samples were eluted using the standard methodology for both antigens (p = <0.0001). For antibody level (optical density), responses were on average 11.3% higher for AMA-1, and 21.4% higher for MSP-142 when using standard rather than combined elution. Linear regression analysis showed that for AMA-1 an increase of titre 1 using combined elution was associated with an increase of titre 1.773 (95% CI 1.712-1.834; p < 0.0001) using standard elution. For MSP-142 an increase of titre 1 using combined elution was associated with an increase of titre 1.811 (95% CI 1.647-1.975, p < 0.0001) using standard elution. For use as marker of exposure, antibody responses against AMA-1 and MSP-142 are commonly combined to give a prevalence of any anti-P. falciparum antibodies [17,18,20]. Between standard and combined elution methods seroprevalence of antibody responses to AMA-1 and MSP-142 did not differ significantly (p >0.8), and for both methods showed a strong age-dependent increase (Figure 2; p <0.0001). Within age-groups, antibody seroprevalence did not differ significantly between elution methods (p >0.5).


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)

Antibody level from standard and dual filter paper blood spot elution methods for AMA-1 and MSP-142. A. Scatter plot showing anti-AMA-1 IgG level detected in 236 individuals by standard (x-axis) and combined (y-axis) elution of filter paper blood spots. R2 (linear regression) = 0.93 (p = <0.0001). B. Scatter plot showing anti-MSP-142 IgG level detected in 236 individuals using standard (x-axis) and combined (y-axis) elution of filter paper blood spots. R2 (linear regression) 0.671 (p = <0.0001).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Antibody level from standard and dual filter paper blood spot elution methods for AMA-1 and MSP-142. A. Scatter plot showing anti-AMA-1 IgG level detected in 236 individuals by standard (x-axis) and combined (y-axis) elution of filter paper blood spots. R2 (linear regression) = 0.93 (p = <0.0001). B. Scatter plot showing anti-MSP-142 IgG level detected in 236 individuals using standard (x-axis) and combined (y-axis) elution of filter paper blood spots. R2 (linear regression) 0.671 (p = <0.0001).
Mentions: Antibodies were eluted from 236 filter papers by both the standard elution procedure and the combined elution procedure. For both AMA-1 and MSP-142, a strong positive correlation was observed between the IgG responses of filter paper blood samples eluted by standard and combined methods (Figure 1). While strongly correlated, there was a tendency toward higher antibody responses when samples were eluted using the standard methodology for both antigens (p = <0.0001). For antibody level (optical density), responses were on average 11.3% higher for AMA-1, and 21.4% higher for MSP-142 when using standard rather than combined elution. Linear regression analysis showed that for AMA-1 an increase of titre 1 using combined elution was associated with an increase of titre 1.773 (95% CI 1.712-1.834; p < 0.0001) using standard elution. For MSP-142 an increase of titre 1 using combined elution was associated with an increase of titre 1.811 (95% CI 1.647-1.975, p < 0.0001) using standard elution. For use as marker of exposure, antibody responses against AMA-1 and MSP-142 are commonly combined to give a prevalence of any anti-P. falciparum antibodies [17,18,20]. Between standard and combined elution methods seroprevalence of antibody responses to AMA-1 and MSP-142 did not differ significantly (p >0.8), and for both methods showed a strong age-dependent increase (Figure 2; p <0.0001). Within age-groups, antibody seroprevalence did not differ significantly between elution methods (p >0.5).

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