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

Abstract

BackgroundInforming 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.MethodsFilter 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.ResultsAntibody 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.ConclusionCombined 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.

Is asymptomatic malaria really asymptomatic? Hematological, vascular and inflammatory effects of asymptomatic malaria parasitemia

“…To determine 129 molecular prevalence of parasites, DNA was extracted from filter papers using Saponin-Chelex 130 extraction. A nested PCR reaction was performed after the data collection was completed at the 131 Radboud university medical center in The Netherlands, amplifying the P. falciparum and vivax 132 specific 18S ribosomal DNA fragment (37,38). A nested PCR was performed, the nest 1 reaction 133 contained Plasmodium genus specific primers.…”

High Levels of Asymptomatic and Subpatent Plasmodium falciparum Parasite Carriage at Health Facilities in an Area of Heterogeneous Malaria Transmission Intensity in the Kenyan Highlands

“…25 Nested PCR (nPCR) was used to test for the presence of parasite DNA to provide a gold standard measure for current infection. A Chelex-saponin approach was used to extract DNA as described by Baidjoe and others, 24 and the nPCR assay targeting the 18S ribosomal subunit of P. falciparum was used as previously described. 26 Samples that were positive by nPCR were then selected for subsequent analysis to identify allelic diversity using the polymorphic MSP2 region to provide an alternate measure of transmission intensity.…”

“…Briefly, two blood spot sections per sample were punched, and antibodies were eluted according to work by Baidjoe and others. 24 Antibody prevalence for each antigen was determined after defining a cutoff optical density (OD) based on a standard curve of known antibody concentration using the mixture model and normalized across plates. 20,25 A person was considered to be seropositive if they had normalized OD values above the cutoff for at least one of the antigens tested.…”

“…26 Samples that were positive by nPCR were then selected for subsequent analysis to identify allelic diversity using the polymorphic MSP2 region to provide an alternate measure of transmission intensity. 24,25,27 An additional nPCR reaction was conducted to amplify the block-3 region of the MSP2 domain targeting the FC27 and IC3D7 allelic variants. 28 The product of the MSP2 PCR was viewed on 1.5% agarose gel to determine the dilution factor necessary to prepare samples for capillary electrophoresis: intense bands were diluted at 1:100, moderate bands were diluted at 1:40, and faint bands were diluted at 1:10.…”

The Effect of Storage and Extraction Methods on Amplification of Plasmodium falciparum DNA from Dried Blood Spots

“…1,2 Although it is appreciated that DNA degrades over time, there have been few systematic efforts to quantify this degradation, and there is a lack of agreed-upon standards for storage, extraction, and amplification of plasmodial DNA from DBS. Studies have shown that humidity negatively effects DNA quality and that Whatman 3MM and 903 filter paper (Whatman, Clifton, NJ) are ideal for long-term storage.…”