Efficient monitoring of the blood-stage infection in a malaria rodent model by the rotating-crystal magneto-optical method

Intense research efforts have been focused on the improvement of the efficiency and sensitivity of malaria diagnostics, especially in resource-limited settings for the detection of asymptomatic infections. Our recently developed magneto-optical (MO) method allows the accurate quantification of malaria pigment crystals (hemozoin) in blood by their magnetically induced rotation. First evaluations of the method using β-hematin crystals and in vitro P. falciparum cultures implied its potential for high-sensitivity malaria diagnosis. To further investigate this potential, here we study the performance of the method in monitoring the in vivo onset and progression of the blood-stage infection in a rodent malaria model. Our results show that the MO method can detect the first generation of intraerythrocytic P. berghei parasites 66–76 hours after sporozoite injection, demonstrating similar sensitivity to Giesma-stained light microscopy and exceeding that of flow cytometric techniques. Magneto-optical measurements performed during and after the treatment of P. berghei infections revealed that both the follow up under treatment and the detection of later reinfections are feasible with this new technique. The present study demonstrates that the MO method – besides being label and reagent-free, automated and rapid – has a high in vivo sensitivity and is ready for in-field evaluation.


Introduction
As malaria remains one of the most serious health and economic burdens of the globe, the study of human malaria is one of the most important agendas of today's scientific research.
The study of the disease involves a myriad of methods ranging from epidemiological analysis and clinical studies to laboratory model systems such as in vitro parasite cultures in human red blood cells (RBCs) and in vivo rodent models. Rodent models of malaria have been widely and successfully used to study the biology and pathology of the malaria infection and host immune response [reviewed in Zuzarte-Luis 2014a], as well as for in vivo evaluation of novel vaccine and drug candidates [Mota 2001, Khan 1991, despite their limitations to replicate certain aspects of the human disease [White 2012, Craig 2008].
In such studies the most commonly used parameter to monitor the progression of the infection is the appearance of parasites in the circulation and the assessment of parasitemia, which is often challenging to rapidly, yet accurately determine.
The reference method of light microscopy of Giemsa-stained thin blood smears is a demanding, labor-intensive procedure, often lacking the desired sensitivity, especially at low yoelii [Ono 2007] and P. berghei strain ANKA [Franke-Fayard 2004], and the luciferin substract used for these assays is costly.
The need for a universally applicable and automated method for blood stage parasite quantification has motivated several investigations of the unique physical properties of malaria pigment (hemozoin-hz). As a result, its utilization as an alternative target of magnetic and/or optical detection of the infection has been proposed by several groups [Rebelo 2012, Karl 2008, Mens 2010, Lukianova-Hleb 2013. Hemozoin is a micro-crystalline heme compound produced by all Plasmodium spp. during the intraerythrocytic stage as they detoxify free heme derived from hemoglobin digestion , Slater 1991, Fulton 1953]. The content of hemozoin increases as the parasite matures, thus constituting an optimal indicator of parasite maturation. Alongside infection diagnostics, the hemozoinbased detection of parasite maturation can be utilized in antimalarial drug development as well. The feasibility and efficiency of the concept has been recently demonstrated in a novel reagent-free drug sensitivity assay based on the flow cytometric detection of hemozoin [Rebelo 2013, Rebeleo 2015b].
As previously described, the rotating-crystal magneto-optical (MO) technique utilizes the linear dichroism and magnetic anisotropy highly specific to the hemozoin crystals to determine their concentration in liquid suspensions [Butykai 2013]. In our recent study the sensitivity of the method has been evaluated using ring and schizont stages from P. falciparum in vitro cultures where detection thresholds of 0.0008% and 0.0002% parasitemia have been found, respectively [Orban 2014]. These sensitivity results supplemented by other advantages of the MO method -label and reagent-free, cost-effective and quick -implied, that it's a suitable candidate for the detection of the infection as a laboratory tool in malaria research or, eventually, as an in-field diagnostic tool. Here the performance of the method in monitoring the onset and progression of the blood stage infection of a malaria mouse model is presented. The aim of the study was to systematically investigate the abovementioned questions in vivo before engaging in complex and expensive field trials. Therefore, to mimic the natural infection under controlled conditions, mice were inoculated with sporozoites and the onset of the blood stage was assessed by different diagnostic tools to compare the MO method's performance to wellknown techniques. The control methods used in the study were light microscopy, real-time PCR analysis and flow cytometry.
MO measurements during successful treatment of mice with P. berghei infections were also performed in order to gain insight into the clearance rate of hemozoin as the viable parasites disappear from the circulation; and to establish a time-frame after which the MO signal in successfully treated mice vanishes.

Animals, parasites and treatment
In two independent experiment series (A and B), four and three BALB/c mice (Charles River, Spain) were infected with the transgenic P. berghei ANKA (259cl2) that constitutively expresses GFP during the whole life cycle. Sporozoites were obtained by disruption of the salivary glands of freshly dissected, infected female Anopheles stephensi mosquitoes and In the treatment measurements (T series) five mice with severe P. berghei ANKA infections were treated by daily administration of 7 mg/ml of chloroquine for seven days. As the mice exhibited anemia on the first day of treatment as a consequence of the advanced infection, no further blood collection was performed until the administration of the third dose of chloroquine (day 2). On this day blood samples from 4 mice were analyzed again by the MO method and by light microscopy. Further blood sampling and analysis was performed on day 4, 5, 7, 10, 12, and 13, as shown in Figure 3. Microscopic observation of thin blood smears indicated that the treatment was successful and parasites have already been cleared from the blood stream on day 4, after the administration of five doses of chloroquine.

Ethics statement
This study was approved by the Ethical Committee of the Faculty of Medicine, University of Lisbon. All experiments involving animals were performed in compliance with the relevant laws and institutional guidelines. Animals were monitored daily and every effort was made to minimize suffering. Upon completion of experiments, mice were euthanized via administration of CO2 followed by cervical dislocation.

Magneto-optical measurements
All reagents were purchased from Sigma Aldrich (St Louis, Mo, USA) unless stated otherwise.
For the magneto-optical measurements 30 ul of blood was transferred from each mouse directly into 570 ul of lysis solution (0.066 V/V% Triton X-100 in 3 mM NaOH). The lysed sample was measured after 5 minutes, whilst the hemozoin crystals were liberated from the RBC's and from the parasites constituting a homogenous crystal suspension. MO measurements were performed using 450 ul from each lysed sample.
The prototype of the MO setup, as well as the underlying physical principles of the detection method, are described in a former study [Butykai 2013]. Briefly, the lysed sample, filled into a cylindrical sample holder, is inserted into the center of an assembly of permanent magnets arranged in a ring, which creates a strong uniform magnetic field (B = 1T) at the sample position and results in the co-alignment of the hemozoin crystals. When the magnetic ring is rotated, the co-aligned hemozoin crystals follow this rotation. During the measurement polarized light from a laser diode is transmitted through the sample in the direction perpendicular to the plane of the rotating magnetic field. The rotation of the co-aligned dichroic crystals gives rise to a periodic change in the transmitted intensity (ΔT), whichdivided by the time-averaged intensity (T) -corresponds to the measured MO signal (ΔT/T in %).
During the measurements described in this study, the rotational speed of the magnet was gradually increased from 1Hz to 50Hz, and the MO signal was recorded at each step. However, it has already been demonstrated that the best signal-to-noise ratio is observed in the range

Assessment of the parasitemia level in the first intraerythrocytic cycle by the MO method
In our previous studies we demonstrated that the MO signal is directly proportional to the be schizonts is ΔT/T = 5.6 ± 2*10 -3 %. According to our previous report [Butykai 2013], this would correspond to a hemozoin concentration of 0.08 ± 0.029 ng/μl in the measured crystal suspension that was produced by a 20-fold dilution of the collected whole blood sample.
Assuming that by the end of their life cycle the schizonts have converted roughly 80% of the host cell's hemoglobin into hemozoin, and using standard hemoglobin and RBC concentration values for BALB/c mice [Russell 1951], the parasitemia level at the end of the first erythrocytic cycle is estimated to be 0.0018 ± 0.0007%. This estimate for the parasitemia level at the end of the first erythrocytic cycle is in good agreement with the first direct light-microscopy count of ~0.15% at 90 hours pi (beginning of the third erythrocytic cycle) using an approximately 8fold multiplication rate upon invasion in accordance with data available in the literature [Janse 2003, Janse 2006].
To support the consistency of the above estimations and the robustness of the hemozoin concentration measurements via the MO method, a reversed calculation can be applied for the MO values measured at 66 hour pi when circulating parasites are assumed to be 10-14 hours old rings, at the previously established ~0.0018% parasitemia level. The averaged MO value of ΔT/T=1.9 ± 0.7*10 -3 % corresponds to a hemozoin concentration of 0.027 ± 0.01 ng/μl. In order to produce this amount of hemozoin parasites have had to convert 25-30% of the host cell's hemoglobin, which is in good agreement with data found in the literature , Weissbuch 2008, Gligorijevic 2006]. This correspondence confirms that the MO signal can be consistently related to the parasitemia level and the course of the infection can be traced via the precise measurement of in vivo hemozoin production.

Monitoring parasite clearance by the MO method
The ability of the MO method to follow parasite clearance during and after treatment, and the persistence of positive test results were also investigated.
In this study 5 mice with severe P. berghei infection were treated by daily administration of chloroquine for seven days as described in the 'Methods' section. The MO signal was measured both during the treatment and for the nine consecutive days after treatment to study i) the correlation between parasite clearance and the magnitude of MO signal and ii) the timescale over which the MO signal is reduced to the level of the uninfected baseline following a successful treatment. was reported for P. falciparum infections [Day 1996] and also with the observations of midand long-term hemozoin kinetics reported for in vivo P. Berghei infections [Frita 2011]. In the latter study 7 days after parasite clearance ~1.7% of the phagocytes were found to contain hemozoin granules.
By day 10 the MO signals in all treated mice decreased to the level of the uninfected baseline.
Indeed, the signal remained at this level for the consecutive two days, confirming the absence of the infection and yielding true negative diagnostic results.

Conclusions
The rotating-crystal magneto-optical detection method has demonstrated excellent sensitivity to detect low concentrations of synthetic hemozoin crystals [Butykai 2013 While studies on field isolates are required for the final evaluation of the method for human infections, we believe that the present study provides a solid basis for the implementation of such in-field tests.