Unified Parasite Lactate Dehydrogenase and Histidine-Rich Protein ELISA for Quantification of Plasmodium falciparum

Abstract

There is a need for more objective and quantitative tools to replace microscopy in malaria diagnosis. Emphasis has recently been placed on alternative methods such as immunochromatography-based rapid tests. However, these tests provide only qualitative results. Two bio-molecules, parasite lactate dehydrogenase (pLDH) and histidine-rich proteins (HRPs), that are released by the intra-erythrocytic stages of the parasite offer certain specific characteristics that could potentially improve malaria diagnosis. In this paper, we describe a protocol for a unified sandwich ELISA that allows for the separate but concurrent measurement of pLDH and HRP biomolecules in aliquots taken from the same samples. Freshly drawn blood from a healthy unexposed adult male was used to serially dilute in vitro cultivated and synchronized ring stage Plasmodium falciparum parasites. Commercially available ELISA formats were modified to allow for the measurement of pLDH and HRP from aliquots of the same samples. The pLDH and HRP levels in the samples spiked with known numbers of infected red blood cells (iRBCs) were measured, and the values were used to generate standard graphs. The standard graphs were used to estimate the numbers of iRBCs in test samples. Serially diluted recombinant proteins were similarly used to generate a calibration curve, allowing for the expression of test results in nanograms of their respective recombinant protein. Levels of pLDH and HRPs were determined by using 1) P. falciparum culture material (cells and medium) 2) P. falciparum infected human blood (N = 6) samples, and 3) plasma from P. falciparum-infected patient (N = 22) samples. The parasite density of all culture and infected patient samples was also estimated by microscopy. Both pLDH and HRP levels correlated positively with the parasite density assessed by microscopy: Pearson correlation coefficient pLDH (r = 0.754, P < 0.0001, 95% CI: 0.47-0.89); HRP (r = 0.552, P < 0.007, 95% CI: 0.16-0.79). The HRPs seem to be released in larger quantities than pLDH (in a ratio of ~1 pLDH:~6 HRP), making the detection of HRP in culture material, blood, and plasma easier. The modified ELISA assay with quantitative measurement of pLDH and HRPs may provide a valuable tool for malaria research and patient management.

Rapid Malaria Tests: Where Do We Go After 20 Years?

“…Studies using commercially available ELISAs estimate that the number of pLDH molecules in intraerythocytic parasites is comparable to the level of HRP-2 proteins, although differences in the sensitivity of the two assays and characterization of the enzyme standards prevented exact comparisons. 44 The comparable levels of antigen indicate that the theoretical limit of pLDH versus HRP-2 detection is comparable, which is consistent with field studies that demonstrate a similar threshold of 100 parasites/µL for reliable detection. It is also important to note that levels of pLDH and HRP-2 measured by ELISA roughly correlate with parasitemia.…”

Genome scanning of Amazonian Plasmodium falciparum shows subtelomeric instability and clindamycin-resistant parasites

“…This variability may contribute to the failure of rapid diagnostic tests that are directed toward antigens such as the histidine-rich protein II, encoded by a subtelomeric region that appears to be deleted in a subpopulation of Peruvian P. falciparum. This deletion has been previously described (Pieroni et al 1998;Bell et al 2005;Wongsrichanalai et al 2007;Martin et al 2009), and the presence of this deletion in the Amazon suggests that widespread use of malaria rapid diagnostic tests that depend on HRP2 will result in missed diagnoses.…”

“…We found deleted regions covering 20-25 kb in subtelomeric regions, including the apparent deletion of important diagnostic loci like histidine-rich proteins II and III (HRP2, MAL7P1.231 and HRP3, MAL13P1.480, respectively) (Supplemental File 2; Supplemental Fig. S1; Wongsrichanalai et al 2007;Martin et al 2009). Because many malaria diagnostic tests that distinguish between P. falciparum and P. vivax are based on immune recognition between commercial antibodies and these proteins (Wongsrichanalai et al 2007), it is likely that false-negative readings could be obtained.…”

Structural basis for discriminatory recognition of Plasmodium lactate dehydrogenase by a DNA aptamer

“…S9). This limit of detection is a promising starting point with regards to future clinical diagnostic applications because patients infected with PfLDH typically show PfLDH plasma levels around 3-15 pg/μL plasma (26), which can easily be concentrated to a range within the limit of detection against a typical background human LDH level of 100-400 pg/μL. Therefore, discrimination by the 2008s aptamer would be sufficient, and the sensitivity should be attainable with further development.…”