Evaluation of three different DNA extraction methods from blood samples collected in dried filter paper in Plasmodium subpatent infections from the Amazon region in Brazil

Abstract

SUMMARYAsymptomatic Plasmodium infection is a new challenge for public health in the American region. The polymerase chain reaction (PCR) is the best method for diagnosing subpatent parasitemias. In endemic areas, blood collection is hampered by geographical distances and deficient transport and storage conditions of the samples. Because DNA extraction from blood collected on filter paper is an efficient method for molecular studies in high parasitemic individuals, we investigated whether the technique could be an alternative for Plasmodium diagnosis among asymptomatic and pauciparasitemic subjects. In this report we compared three different methods (Chelex®-saponin, methanol and TRIS-EDTA) of DNA extraction from blood collected on filter paper from asymptomatic Plasmodium-infected individuals. Polymerase chain reaction assays for detection of Plasmodium species showed the best results when the Chelex®-saponin method was used. Even though the sensitivity of detection was approximately 66% and 31% for P. falciparum and P. vivax, respectively, this method did not show the effectiveness in DNA extraction required for molecular diagnosis of Plasmodium. The development of better methods for extracting DNA from blood collected on filter paper is important for the diagnosis of subpatent malarial infections in remote areas and would contribute to establishing the epidemiology of this form of infection.

Effectiveness of a Malaria Surveillance Strategy Based on Active Case Detection during High Transmission Season in the Peruvian Amazon

“…Given the limitations of molecular tests in the detection of very low parasitaemia using blood spot filter papers [53], all qPCR results in ACD visits were taken into account to characterize malaria infections in the participants. In this regard, the detection of mixed/co-infections posed a major challenge since molecular tests like qPCR frequently identify only one species (the species with the most abundant parasite DNA) [54].…”

Towards a re-emergence of chloroquine sensitivity in Côte d’Ivoire?

“…Plasmodial DNA was extracted with methanol from DBS cut into small pieces were immersed in 1 mL of washing buffer (950 μL of 1× PBS plus 50 μL of 10% saponin) and incubated overnight at 4 °C [ 17 ]. The wash buffer was removed and 150 μL of methanol were added.…”

Dried Blood Spots for Global Health Diagnostics and Surveillance: Opportunities and Challenges

“…The recovery and stability of malaria-related histidine-rich protein 2 (HRP2) 99 and mRNA were also reported to be dependent on the type of DBS. 100 , 101 In a report by Miguel et al, 102 none of the three commercially available reagents were able to reliably extract DNA associated with Plasmodium falciparum or Plasmodium vivax infection from blood stored on cotton-based filter paper, although others have shown a dependence on the type of DBS. 41 , 103 The use of cards designed to preserve nucleic acids was found to provide sufficient stability for detecting single-species malaria infection but failed to diagnose individuals with mixed P. vivax/falciparum infections.…”