De et al. Malar J (2016) 15:3 DOI 10.1186/s12936-015-1027-2 Malaria Journal METHODOLOGY Open Access Plasmodium berghei bio‑burden correlates with parasite lactate dehydrogenase: application to murine Plasmodium diagnostics Sai Lata De1, Danielle I. Stanisic1, Fabian Rivera2, Michael R. Batzloff1, Christian Engwerda2 and Michael F. Good1* 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 differ- ent 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 Plasmo- dium-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 infec- tions. Unlike existing methods, it permits the estimation of both circulating and sequestered parasites, allowing a more accurate assessment of parasite bio-burden. Keywords: Malaria, pLDH, Murine Plasmodium, Parasite bio-burden Background labelling [11] have been used to measure parasitaemia in Malaria continues to be a leading cause of mortality in in vivo preclinical efficacy studies for anti-malaria drugs endemic countries, despite control and eradication initia- and vaccine candidates. However, these approaches are tives, such as the use of insecticide-treated bed nets and limited to detecting circulating parasites and do not anti-malaria drug treatment [1]. Clinical disease typically account for sequestered parasites, which is important for presents as fever, chills, severe headache, and vomiting, determining total parasite bio-burden. Bio-luminescence and requires a prompt diagnosis followed by effective imaging, using transgenic parasite that express the fire- treatment to ensure resolution of the infection. Impor- fly luciferase reporter protein, has been used not only tantly, disease outcomes are directly correlated with total to measure parasite growth in vivo and in vitro [10], but parasite burden [2]. In rodent malaria models, Giemsa- also to visualize in vivo the parasite sequestration in deep stained blood films (the gold-standard microscopy tech- tissues [13–16]. nique) [3, 4], PCR [5], flow cytometry [6, 7], fluorescent Various assays using specific antibodies to parasite [8, 9] or DNA/RNA [10–12] markers and radioisotope antigens have been employed [17–24]. Immunochro- matographic assays, which utilize monoclonal antibod- ies against specific Plasmodium enzymes, are under *Correspondence: [email protected] development. The most promising antigens explored 1 Institute for Glycomics, Griffith University, Gold Coast, QLD, Australia so far include: histidine rich protein-2 (HRP-2) [25], Full list of author information is available at the end of the article © 2015 De et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/ zero/1.0/) applies to the data made available in this article, unless otherwise stated. De et al. Malar J (2016) 15:3 Page 2 of 11 parasite-specific lactate dehydrogenase (pLDH) [18, 19, during murine malaria infections. This assay could be 26–28], and aldolase [29, 30]. These enzymes are involved established as an alternative approach to measure para- in metabolic pathways essential for the growth and sur- site bio-burden in efficacy studies. vival of Plasmodium parasites [29]. The enzyme pLDH is a soluble, energy-producing Methods enzyme that is involved in the last step of the glycolytic Mice and ethics statement pathway [29]. As the red blood cells do not have func- Female BALB/c mice aged 4–6 weeks were purchased tional mitochondria and the parasites have minimum from the Animal Resource Centre (ARC) (Canning Vale, oxygen uptake for the citric acid cycle [31], it is highly Perth, Australia) and maintained under appropriate ARC dependent on anaerobic glucose metabolism [32, 33]. and Griffith University conditions. This study was -car pLDH is produced by both asexual blood-stage para- ried out in strict accordance with the National Health sites as well as the sexual stages, with a larger quantity and Medical Research Council of Australia guidelines, as of pLDH being produced during the asexual stage [29]. detailed in the document, Australian Code of Practice for pLDH antigen is preferable as a diagnostic marker over the Care and Use of Animals for Scientific Purposes, 8th other antigens such as HRP-2, which is limited to Plas- ed [46]. The Griffith University Animal Ethics Commit- modium falciparum only [34]. Moreover, some P. falcipa- tee (GLY/05/12/AEC) and the QIMR Berghofer Medi- rum strains have a deletion in the HRP-2 gene, resulting cal Research Institute Ethics Committee (A02633M) in false negative tests [35]. Unlike HRP-2, pLDH does not approved the relevant animal procedures and protocols. persist in the blood [36, 37] and is cleared immediately post-active infection [18–20, 22, 38, 39], thus making Parasites and infections pLDH an ideal marker to estimate parasite bio-burden at Cloned lines of P. chabaudi AS, P. yoelii 17XNL, P. yoe- the time of the assay. lii YM, P. berghei and P. vinckei were used (provided by Previously, monoclonal antibodies specific for pLDH Richard Carter, University of Edinburgh, UK). Stabilates have been used to determine the sensitivity of Plas- were maintained by intra-venous (IV) and intra-peri- modium berghei to anti-malarial drugs in vitro [40]. A toneal (IP) passaging of 106 parasitized red blood cells chromogenic pLDH assay has also been employed to (pRBC) into naïve BALB/c mice. Plasmodium berghei enumerate the parasites in the blood of mice challenged ANKA luc lines (provided by Chris Janse, Leiden Univer- with Plasmodium yoelii 17XNL post vaccination with sity Medical Centre, The Netherlands) were used in the MSP1-19 [41]. However, none of these approaches was bio-luminescent experiments for in vivo imaging after compared to an established assay to quantify and validate one in vivo passage in mice. total parasite bio-burden. The pLDH amino acid sequence has a 90 % sequence Evaluation of parasitaemia by microscopy identity amongst all human Plasmodium species [33, 42]. Thin blood smears were prepared, air dried, fixed in For human parasites, monoclonal antibodies against the methanol and stained with Giemsa. Slides were exam- shared common epitopes can be used to detect all species ined using bright field microscopy and counts were per- [43, 44]. Genetic conservation and variation of pLDH formed with 100× magnification. For negative or low across different human and rodent species and strains of parasitaemia films (<1 %), at least 20 fields on the slide Plasmodium was reported by Talman et al. [45]. Nucle- were counted. For high parasitaemia, at least 500 RBCs otide BLAST analysis using 951 nucleotides of the P. were counted. Percent parasitaemia was calculated as the falciparum 3D7 (Pf LDH) gene coding sequence [Acces- number of pRBCs multiplied by 100 divided by the num- sion ID XM_001349953.1] as the reference revealed ber of total RBCs. the following per cent identity in different species of murine Plasmodium: 86 % identity with P. yoelii 17XNL Assessment of pLDH antigen production in murine malaria [Accession ID XM_719008.1]; 85 % with Plasmodium models chabaudi chabaudi [Accession ID XM_740087.1]; 85 % Qualitative and quantitative levels of pLDH antigens with P. berghei ANKA [Accession ID XM_674309.1]; in BALB/c mice infected with P. chabaudi AS, P. yoelii and 83 % with Plasmodium vinckei [Accession ID 17XNL, P. yoelii YM, P. berghei ANKA, and P. vinckei XM_008624100.1]. The high degree of sequence similar- were determined using the pLDH SD Malaria Antigen ity could potentially be exploited for use in diagnostics ELISA kit (Standard Diagnostics), a sandwich ELISA for for rodent malaria parasites (Table 1). the qualitative detection of human Plasmodium pLDH. This study investigated the use of a commercial human For quantitative assessment, blood was collected on the Plasmodium pLDH ELISA diagnostic kit for detect- following days for each of the rodent malaria parasites: ing pLDH antigen as a measure of parasite bio-burden P. chabaudi AS: Day 7 (29 %), P. berghei ANKA: Day 4 De et al. Malar J (2016) 15:3 Page 3 of 11 Table 1 pLDH protein sequence alignment analysis