Plasmodium falciparum HRP2 ELISA for analysis of dried blood spot samples in rural Zambia

Abstract

BackgroundDried blood spots are commonly used for sample collection in clinical and non-clinical settings. This method is simple, and biomolecules in the samples remain stable for months at room temperature. In the field, blood samples for the study and diagnosis of malaria are often collected on dried blood spot cards, so development of a biomarker extraction and analysis method is needed.MethodsA simple extraction procedure for the malarial biomarker Plasmodium falciparum histidine-rich protein 2 (HRP2) from dried blood spots was optimized to achieve maximum extraction efficiency. This method was used to assess the stability of HRP2 in dried blood spots. Furthermore, 328 patient samples made available from rural Zambia were analysed for HRP2 using the developed method. These samples were collected at the initial administration of artemisinin-based combination therapy and at several points following treatment.ResultsAn average extraction efficiency of 70% HRP2 with a low picomolar detection limit was achieved. In specific storage conditions HRP2 was found to be stable in dried blood spots for at least 6 months. Analysis of patient samples showed the method to have a sensitivity of 94% and a specificity of 89% when compared with microscopy, and trends in HRP2 clearance after treatment were observed.ConclusionsThe dried blood spot ELISA for HRP2 was found to be sensitive, specific and accurate. The method was effectively used to assess biomarker clearance characteristics in patient samples, which prove it to be ideal for gaining further insight into the disease and epidemiological applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-017-1996-4) contains supplementary material, which is available to authorized users.

Characterization of Plasmodium Lactate Dehydrogenase and Histidine-Rich Protein 2 Clearance Patterns via Rapid On-Bead Detection from a Single Dried Blood Spot

“…Optimum conditions for HRP2 recovery from DBS were previously reported. 16 To determine whether this method achieved sufficient elution of p LDH, the recoveries of both biomarkers were compared across multiple extraction times in PBST. The p LDH extraction efficiencies were not significantly different from those of HRP2 across all DBS extraction incubation times.…”

“…The p LDH and HRP2 concentrations in the 18,450 parasite/μL stock were previously reported as 1.3 and 1.7 pM per parasite/μL, respectively. 16 , 19 In addition, HRP2 in the 43,600 parasites/μL stock was previously determined to be 2.2 pM per parasite/μL. 23 The p LDH concentration of in-house D6 P. falciparum culture (stock 43,600 parasites/μL) was determined to be 4.4 pM per parasite/μL using a standard well-plate ELISA, N = 6 ( Supplemental Text 1 ).…”

“…Dried blood spot extraction was adapted from a previously reported method. 16 Dried blood spot was prepared by depositing 10 μL of parasitized whole blood onto Whatman 903 protein saver cards. The spots were allowed to air-dry for a minimum of 4 hours, removed using a 6-mm biopsy punch, and placed in 2-mL microcentrifuge tubes (one spot per tube).…”

“…Recently, two studies have measured Plasmodium falciparum histidine-rich protein 2 (HRP2), the primary protein biomarker used to diagnose malaria, in DBS patient samples. Rogier et al 15 used their DBS detection method to evaluate the accuracy of HRP2-based rapid diagnostic tests, and Gibson et al 16 demonstrated the persistence of HRP2 in patient DBS samples after treatment compared with microscopy. Although both of these studies demonstrated sensitive HRP2 quantitation from DBS, there are several disadvantages of using HRP2 alone as a diagnostic marker for malaria.…”

Plasmodium falciparum Parasitemia and Band Sensitivity of the SD Bioline Malaria Ag P.f/Pan Rapid Diagnostic Test in Madagascar

“…1 On the other hand, there is only a very limited correlation between parasitemia and PfHRP2 level, at best, in clinical samples. [34][35][36][37][38] Because there are similar ranges of concentrations for PfHRP2 and pLDH antigens within P. falciparum-infected blood samples, with a significant positive correlation between the two, it may be that the difference in antibody-binding avidity between PfHRP2 (multiple binding epitopes) and pLDH (single epitope) is a reason for the observed differences in positivity and intensity of the respective test bands. 13 The PfHRP2 and pLDH antigen concentrations were not measured in our study, which may be a limitation to explain how high parasitemia influences the sensitivity of the different test bands.…”