Enzyme-Linked Immunosorbent Assay for Detection of Plasmodium falciparum Histidine-Rich Protein 2 in Blood, Plasma, and Serum

Abstract

Microscopy, the gold standard for the detection and quantification of malaria parasites in blood, is in many aspects deficient for this purpose. The method is poorly reproducible and can be inaccurate because Plasmodium falciparum parasites sequester for a portion of each asexual cycle. Due to these deficiencies, biomarkers such as P. falciparum histidine-rich protein 2 (PfHRP2) are increasingly being used. In this study, we evaluated the use of a commercial PfHRP2 enzyme-linked immunosorbent assay (ELISA) kit with some procedural modifications. We determined the linear range of the assay, including the lower limits of detection and quantitation, using recombinant PfHRP2 (rPfHRP2). In 10 repeat experiments, the linear range of optical densities (ODs) at 450 to 650 nm was from 0.05 ؎ 0.002 to 2.28 ؎ 0.042, corresponding to 3.91 to 250 ng/ml of rPfHRP2. The coefficient of variation (CV) at each target concentration ranged from 1.93 to 8.07%. Using cultured parasites, we confirmed the linear range of ODs as well as the association between the PfHRP2 ELISA results and the microscopic parasite densities. For whole-blood samples spiked with cultured, washed, ring-stage-infected red blood cells (iRBCs), the linear range was 11.7 to 750 iRBCs/l, with CVs of 0.29 to 7.56%. The same spiked samples evaluated by microscopists had similar sensitivities, but the CVs were unacceptably high (20.7 to 161.6%). Stock rPfHRP2 was stable through four freeze-thaw cycles (P < 0.05; paired t test). When different patient sample types at different concentrations within the linear range of the assay are compared, the recoveries of PfHRP2 from blood and serum were within ؎20%, whereas the recoveries from plasma ranged between ؉35 and ؊41%. We conclude that PfHRP2 ELISA using whole-blood and serum samples is a suitable adjunct to microscopy and could ultimately benefit malaria intervention trials.Efficacy assessment of malaria intervention studies still relies on microscopy for quantitation of malaria parasites in blood, despite increasing evidence that its reliability is questionable (1,10,18,21,22). The major attributes of malaria parasite microscopy are its cost effectiveness and simplicity, which in resource-poor countries are important considerations. The major disadvantages of microscopy for Plasmodium falciparum parasite quantitation include poor reproducibility, variable sensitivity, and unacceptably high false-positive rates. In addition, the sequestration of parasites for a portion of each asexual cycle makes mature trophozoite and schizont stages unavailable in the peripheral circulation (9).The parasite biomarkers of choice for quantitative estimates of the burden of infection would be those that are detectable in whole blood or in its separated components, i.e., serum and plasma, irrespective of the location of the parasite. Good candidates are histidine-rich protein 2 (HRP2), found only in P. falciparum, and glycolytic lactate dehydrogenase (LDH) and Plasmodium aldolase, both of which are found in all Plasmodium species (19)....

In vitro activity of Pheroid vesicles containing antibiotics against Plasmodium falciparum

“…The assay proved to be sensitive at all parasitemia tested as indicated by the linearity of the fitted curve. This differed from results were obtained by Kifude et al,42 who demonstrated that the lower level of quantification; that is, the point at which the analysis is feasible, was 3.19 ng ml À1 , corresponding to an OD of 0.05. Linearity in our assay was obtained at all parasitemia tested and the lower limit of quantification was 2.18 ng ml À1 corresponding to an OD of 0.04.…”

Correlation between Serum and Plasma Antibody Titers to Mycobacterial Antigens

“…Also, the strong correlations did not appear to be different between lower and higher Ab levels. This is an important observation because prior studies concluded that the significant differences observed between serum and plasma concentrations for certain proteins, such as some cytokines or a plasmodial protein, could have been due in part to the overall low concentrations of these proteins (7,11). Another study that reported significant differences in the detection of microglobulin levels between serum and plasma samples suggested that the differences observed could have been due to a nonspecific effect of heparin or citrate on the microglobulin because they were not observed between serum and EDTA plasma (5).…”

“…For example, some proteins, such as beta-2-microglobulin or Plasmodium falciparum histidine-rich protein 2, have been detected in significantly lower concentrations in human plasma than in serum (5,11), and while very high and significant correlations between plasma and serum levels were obtained for C-reactive protein or insulin (4,7), no statistically significant correlation was found for cytokines such as interleukin-1␤ (IL-1␤) and tumor necrosis factor alpha (TNF-␣) (7). It is also conceivable that storage of plasma samples, which can lead to precipitation of some proteins due to polymerization of fibrin, could result in Ab levels different from those found in serum.…”

Universal mobile electrochemical detector designed for use in resource-limited applications

“…Chronoamperometric measurements (Fig. 3D) performed by the uMED show a linear response for concentrations of PfHRP2 in the clinically relevant range of 0-150 ng/mL (30). In this proof-of-principle experiment, the limit of detection was 20 ng/mL.…”