Plasmodium berghei bio-burden correlates with parasite lactate dehydrogenase: application to murine Plasmodium diagnostics

Abstract

Background: The spectrum of techniques to detect malaria parasites in whole blood is limited to measuring parasites in circulation. One approach that is currently used to enumerate total parasite bio-burden involves the use of bio-luminescent parasites. As an alternative approach, this study describes the use of a commercial ELISA human parasite lactate dehydrogenase (pLDH) detection kit to estimate total parasite bio-burden in murine malaria models.

Methods: The cross reactivity of pLDH in a commercial human malaria pLDH diagnostic kit was established in different components of blood for different murine malaria models. The use of pLDH as a measure of parasite bio-burden was evaluated by examining pLDH in relation to peripheral blood parasitaemia as determined by microscopy and calculating total parasite bio-burden using a bio-luminescent Plasmodium berghei ANKA luciferase parasite.

Results: The pLDH antigen was detected in all four murine Plasmodium species and in all components of Plasmodium-infected blood. A significant correlation (r = 0.6922, P value <0.0001) was observed between total parasite bio-burden, measured as log average radiance, and concentration of pLDH units.

Conclusions: This high throughput assay is a suitable measure of total parasite bio-burden in murine malaria infections. Unlike existing methods, it permits the estimation of both circulating and sequestered parasites, allowing a more accurate assessment of parasite bio-burden.

Fig. 1

a Qualitative detection of pLDH units in different blood components (undiluted) from a mouse infected with Plasmodium chabaudi AS. Individual bars represent negative and positive control from the kit, whole blood obtained from a naïve or P. chabaudi AS-infected BALB/c mouse, serum obtained from a naïve or P. chabaudi AS-infected BALB/c mouse or whole blood obtained from a P. chabaudi AS-infected mouse which had been drug treated. b Representation of a non-linear standard curve to quantify pLDH units (ng/mL). Recombinant pLDH from the kit was serially diluted in both PBS and blood and plotted as log 10 concentration of recombinant pLDH vs the OD. The dotted line shows the linear range of the curve. Each sample was tested in triplicate. Error bars represent the mean ± SE of experimental replicates. ****P value <0.0001, ns not significant. One-way ANOVA was used to determine the statistical significance between groups

Fig. 2

a Quantitative detection of pLDH units in whole blood of BALB/c mice infected with different murine Plasmodium species and b the mean concentration of pLDH units in different components of blood from BALB/c mice infected with Plasmodium chabaudi AS (29.7 %) and Plasmodium yoelii 17XNL (25 %). Percentages indicate the parasitaemia of the mice as determined by microscopy. Each sample was tested in triplicate. Error bars represent mean ± SE of experimental replicates. ****P value <0.0001, **P value <0.01, *P value <0.1, ns not significant. One-way ANOVA was used to determine the statistically significant difference between groups

Fig. 3

Limit of detection (LOD) of pLDH in BALB/c mice infected with a Plasmodium chabaudi AS (24 % parasitaemia) and b Plasmodium berghei ANKA (34 % parasitaemia). For this assay, infected whole blood was serially diluted in naïve mouse blood. ANOVA was used to estimate LOD. Each sample was tested in triplicate. Error bars represent mean ± SE of experimental replicates. The dotted line shows the limit of detection

Fig. 4

Correlation between the concentration of pLDH units and peripheral blood parasitaemia of BALB/c mice (n = 20) infected with Plasmodium chabaudi AS. a Mean parasitaemia in P. chabaudi AS infected mice. b Mean concentration of pLDH units in whole blood obtained from P. chabaudi AS infected mice. c Correlation between the mean concentration of pLDH and peripheral blood parasitaemia (r = 0.9889). Plus symbol indicates that the mice were culled. Error bars represent mean ± SE of biological replicates

Fig. 5

Visualization of luciferase-expressing Plasmodium berghei ANKA parasites in representative BALB/c mice. The distribution of parasites was visualized by measuring luciferase activity in animals by using an I-CCD video camera. Rainbow images show the relative level of luciferase activity ranging from low (blue), to medium (green), to high (yellow, red). The scale of total photon counts can be different between separate illustrations. The white arrows indicate a naïve mouse

Fig. 6

Relationship between pLDH units and total parasite bio-burden in mice (n = 16) infected with Plasmodium berghei ANKA luciferase. a Bio-luminescence measured as average radiance [p/s/cmA2/sr] plotted on a logarithmic scale vs days post infection. b Mean concentration of pLDH units in whole blood obtained from infected mice days post infection. c Mean peripheral blood parasitaemia curves in infected mice as determined by microscopy. d Correlation between the pLDH units and mean parasitaemia, showing the coefficient correlation (r = 0.2445). e Correlation between the pLDH units and log average radiance, showing the coefficient correlation (r = 0.6992). Error bars represent mean ± SE of biological replicates. At day 1, the OD values for the infected mice were less than the control (naïve mouse blood)